Stable crystal of 4-oxoquinoline compound

ABSTRACT

Provision of a stabilized crystal of 6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (compound A). A crystal of compound A, which shows a particular X-ray powder diffraction pattern of a characteristic diffraction peaks at diffraction angles 2θ(°) as measured by X-ray powder diffractometry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(a)to JP 2004-150979 filed May 20, 2004, which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a stable crystal of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid

(hereinafter sometimes to be compound A) and a mixed crystal thereof.The present invention also relates to a pharmaceutical compositioncomprising the crystal or the mixed crystal.

BACKGROUND ART

The present Applicant has disclosed in Japanese Patent Application No.2003-293117 filed by the same Applicant that the above-mentionedcompound A has an inhibitory action on integrase that is an en essentialenzyme for the growth of HIV (Human Immunodeficiency Virus), which is acausative virus of AIDS (Acquired Immunodeficiency Syndrome), and showsan anti-HIV effect (particularly Example 4-32 and Experimental Example).

In general, when a compound is used as a pharmaceutical product,chemical and physical stability of the compound is required so as tomaintain quality and/or facilitate preservation. Not only the finalpharmaceutical composition but also a compound as a synthetic startingmaterial is desirably chemically and physically stable for the samereasons.

Therefore, such compound is preferably a crystal, particularlypreferably a stable crystal. When the compound has crystal polymorphism,the most stable crystal is generally selected.

While the above-mentioned application describes compound A, no concretedescription relating to the crystal form of compound A is found.

SUMMARY OF THE INVENTION

Thus, the present inventors have studied various crystal forms ofcompound A in an attempt to find a stable crystal of compound A. As aresult, they have found that compound A has crystal polymorphism, and acrystal of compound A having a particular crystal form is useful as astable crystal, and based on which findings, they have completed thepresent invention.

Accordingly, one or more embodiments of the present invention providethe following.

[1] A crystal (crystal form II) of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid, which has an X-ray powder diffraction pattern havingcharacteristic diffraction peaks at diffraction angles 2θ(°) of 6.56,13.20, 19.86, 20.84, 21.22, 25.22° as measured by X-ray powderdiffractometer;

[2] a crystal (crystal form III) of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid, which has an X-ray powder diffraction pattern havingcharacteristic diffraction peaks at diffraction angles 2θ(°) of 8.54,14.02, 15.68, 17.06, 17.24, 24.16, 25.74° as measured by X-ray powderdiffractometer;

[3] a crystal (crystal form III) of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid, having an extrapolated onset temperature of 162.1±5.0° C.;

[4] the crystal of any of the above-mentioned [1] to [3], which has apurity of crystal of not less than 70%;

[5] a mixed crystal comprising the crystal of the above-mentioned 1 andthe crystal of the above-mentioned [2] or [3];

[6] the mixed crystal of the above-mentioned [5], wherein the purity ofcrystal is not less than 701;

[7] a pharmaceutical composition comprising the crystal of any of theabove-mentioned [1] to [4] or the mixed crystal of the above-mentioned[5] or [6], and a pharmaceutically acceptable carrier;

[8] an integrase inhibitor comprising the crystal of any of theabove-mentioned [1] to [4] or the mixed crystal of the above-mentioned[5] or [6] as an active ingredient;

[9] an antivirus agent comprising the crystal of any of theabove-mentioned [1] to [4] or the mixed crystal of the above-mentioned[5] or [6] as an active ingredient;

[10] an anti-HIV agent comprising the crystal of any of theabove-mentioned [1] to [4] or the mixed crystal of the above-mentioned[5] or [6] as an active ingredient;

[11] an anti-HIV composition comprising the crystal of any of theabove-mentioned [1] to [4] or the mixed crystal of the above-mentioned[5] or [6] and one or more kinds of other anti-HIV active substances asactive ingredients; and [12] an anti-HIV agent for a multiple drugtherapy with other anti-HIV agent, which comprises the crystal of any ofthe above-mentioned [1] to [4] or the mixed crystal of theabove-mentioned [5] or [6] as an active ingredient.

The crystal or mixed crystal of compound A of the present invention hasthe above-mentioned particular crystal form and is superior in physicaland chemical stability, which in turn has advantage that maintenance ofthe quality of compound A for a long-term becomes possible, whichfacilitates preservation. In addition, they have advantage that handlingduring production of various pharmaceutical compositions and bulk iseasy, which reduces the production cost.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in detail in the following.

The term “crystal form II” of compound A means a crystal of compound A,which has an X-ray powder diffraction pattern having characteristicdiffraction peaks at diffraction angles 2θ(°) of 6.56, 13.20, 19.86,20.84, 21.22, 25.22° as measured by X-ray powder diffractometer.

The term “crystal form III” of compound A means a crystal of compound A,which has an X-ray powder diffraction pattern having characteristicdiffraction peaks at diffraction angles 2θ(°) of 8.54, 14.02, 15.68,17.06, 17.24, 24.16, 25.74° as measured by X-ray powder diffractometer.

The diffraction peak value at the above-mentioned diffraction angle2θ(°) may show slight measurement error due to the measurementinstruments or measurement conditions and the like. To be specific, themeasurement error may be within the range of about ±0.2, preferablyabout ±0.1, more preferably about ±0.06.

In an embodiment, crystal of compound A of the present invention is alsocharacterized by thermal analysis. For example, when the crystal formIII of compound A of the present invention is subjected to DifferentialScanning Calorimetry (DSC), the enthalpy of endothermic peak is about 81J/g, and extrapolated onset temperature is 162.1±5.0° C., preferably162.1±3.0° C., more preferably 162.1±1.0° C., wherein the “extrapolatedonset temperature” means, as defined by JIS K 7121 (measurement methodof transfer temperature of plastic), the temperature at an intersectionof the extrapolated baseline of the lower temperature side toward thehigher temperature side with the tangent line drawn at the point showingthe greatest slope on the leading edge of the melting peak on a lowertemperature side in a DSC curve. When the enthalpy and extrapolatedonset temperature of the endothermic peak is within the above-mentionedrange, the crystal of compound A is stable.

In an embodiment, the crystal of compound A of the present invention maybe either a crystal form II or a crystal form III, or a mixed crystal ofa crystal form II and a crystal form III. For use in a pharmaceuticalproduct of compound A and the like, the crystal form II or crystal formIII is preferable because they are stable crystals, and a crystal formIII is more preferable because it is the most stable crystal. Inaddition, a crystal form II is preferable in view of the absorbabilityby living organisms upon administration as a pharmaceutical composition.

The term “purity of crystal” means the purity of the crystal form II orcrystal form III of compound A. In the case of a mixed crystal of acrystal form II and a crystal form III, it means the ratio of crystalrelative to the total amount of substance of a crystal form II and acrystal form III. The purity of the crystal of the present invention canbe determined by, for example, known methods such as X-ray powderdiffractometry, thermal analysis, and the like. The purity of thecrystal or mixed crystal of the present invention does not need to be100%, and may be not less than 70%, preferably not less than 80%, morepreferably not less than 90%, more preferably not less than 95%, morepreferably not less than 98% Purity within this range is preferable forguaranteeing the quality.

The crystal or mixed crystal of compound A of the present invention canbe administered to a mammal (human, mouse, rat, hamster, rabbit, cat,dog, bovine, sheep, monkey, and the like), and the like as variouspharmaceutical compositions such as anti-HIV agents, HIV integraseinhibitors, antivirus agents, and the like used for, for example, theprophylaxis and/or treatment of AIDS.

When the crystal or mixed crystal of compound A of the present inventionis used as a pharmaceutical composition, it is admixed withpharmaceutically acceptable carriers, excipients, diluents, extendingagents, disintegrants, stabilizers, preservatives, buffers, emulsifiers,flavoring agents, coloring agents, sweetening agents, thickeners,correctives, dissolution aids, and other additives, that are generallyknown, such as as water, vegetable oil, alcohol (e.g., ethanol or benzylalcohol, or the like), polyethylene glycol, glycerol triacetate,gelatin, carbohydrate (e.g., lactose, starch, and the like), magnesiumstearate, talc, lanolin, petrolatum, and the like, formed into tablet,pill, powder, granule, suppository, injection, eye drop, liquid,capsule, troche, aerosol, elixir, suspension, emulsion, syrup, and thelike by a conventional method, and administered systemically ortopically, and orally or parenterally.

While the dose varies depending on age, body weight, symptom, treatmenteffect, administration method, and the like, it is generally from about0.01 mg to about 1 g per administration for an adult, which is givenonce to several times a day orally or in a dosage form of an injectionsuch as intravenous injection and the like.

An anti-HIV agent is generally required to sustain its effect for a longtime, so that it can be effective not only for temporal suppression ofviral growth but also for the prohibition of viral re-growth. This meansthat a prolonged administration is necessary and that a high single dosemay be frequently inevitable to sustain the effect for a longer periodduring night and the like. Such prolonged and high dose administrationincreases the risk of side effects.

In view of this, one of the preferable modes of the present invention issuch compound permitting high absorption by oral administration, andsuch compound capable of maintaining blood concentration of theadministered compound for an extended period of time.

By the “prophylaxis of AIDS” is meant, for example, administration of apharmaceutical agent to an individual who tested HIV positive but hasnot yet developed the disease state of AIDS, administration of apharmaceutical agent to an individual who shows an improved diseasestate of AIDS after treatment but who carries HIV still to be eradicatedand whose relapse of AIDS is worried, and administration of apharmaceutical agent out of a fear of possible infection.

The anti-HIV composition of the present invention is used for, forexample, a multiple drug combination therapy of AIDS. Examples of the“other anti-HIV active substance” to be used for the anti-HIVcomposition include an anti-HIV antibody, an HIV vaccine,immunostimulants such as interferon, and the like, an HIV ribozyme, anHIV antisense drug, an HIV reverse transcriptase inhibitor, an HIVprotease inhibitor, an inhibitor of bond between a bond receptor (CD4,CXCR4, CCR5, and the like) of a host cell recognized by virus and thevirus, and the like.

Specific examples of the HIV reverse transcriptase inhibitor includeRetrovir® (zidovudine), Epivir® (lamivudine), Zerit® (sanilvudine),Videx® (didanosine), Hivid® (zalcitabine), Ziagen® (abacavir sulfate),Viramune® (nevirapine), Stocrin® (efavirenz), Rescriptor® (delavirdinemesylate), Combivir® (zidovudine+lamivudine), Trizivir® (abacavirsulfate+lamivudine+zidovudine), Coactinon® (emivirine), Phosphonovir®Coviracil®, alovudine (3′-fluoro-3′-deoxythymidine), Thiovir(thiophosphonoformic acid), Capravirin(5-[(3,5-dichlorophenyl)thio]-4-isopropyl-1-(4-pyridylmethyl)imidazole-2-methanol carbamic acid), Tenofovir disoproxil fumarate((R)-[[2-(6-amino-9H-purin-9-yl)-1-methylethoxy]methyl]phosphonic acidbis(isopropoxycarbonyloxymethyl) ester fumarate), DPC-083((4S)-6-chloro-4-[(1E)-cyclopropylethenyl]-3,4-dihydro-4-trifluoromethyl-2(1H)-quinazolinone), DPC-961((4S)-6-chloro-4-(cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone),DAPD ((−)-β-D-2,6-diaminopurine dioxolane), Imnunocal, MSK-055, MSA-254,MSH-143, NV-01, TMC-120, DPC-817, GS-7340, TMC-125, SPD-754, D-A4FC,capravirine, UC-781, emtricitabine, alovudine, Phosphazid, UC-781,BCH-10618, DPC-083, Etravirine, BCH-13520, MIV-210, Abacavirsulfate/lamivudine, GS-7340, GW-5634, GW-695634, and the like, wherein(R) means a registered trademark (hereinafter the same) and the names ofother pharmaceutical agents are general names.

Specific examples of the HIV protease inhibitor include a Crixivan®(indinavir sulfate ethanolate), saquinavir, Invirase® (saquinavirmesylate), Norvir® (ritonavir), Viracept® (nelfinavir mesylate),lopinavir, Prozei® (amprenavir), Kaletra® (ritonavir+lopinavir),mozenavir dimesylate([4R-(4α,5α,6β)]-1,3-bis[(3-aminophenyl)methyl]-hexahydro-5,6-dihydroxy-4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-onedimethanesulfonate), tipranavir(3′-[(1R)-1-[(6R)-5,6-dihydro-4-hydroxy-2-oxo-6-phenylethyl-6-propyl-2H-pyran-3-yl]propyl]-5-(trifluoromethyl)-2-pyridinesulfonamide),lasinavir (N-[5 (S)-(tert-butoxycarbonylamino)-4 (S)-hydroxy-6-phenyl-2(R)-(2,3,4-trimethoxybenzyl)hexanoyl]-L-valine 2-methoxyethylenamide),KNI-272((R)—N-tert-butyl-3-[(2S,3S)-2-hydroxy-3-N—[(R)-2-N-(isoquinolin-5-yloxyacetyl)amino-3-methylthiopropanoyl]amino-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide),GW-433908, TMC-126, DPC-681, so buckminsterfullerene, MK-944A (MK944(N-(2 (R)-hydroxy-1(S)-indanyl)-2 (R)-phenylmethyl-4(S)-hydroxy-5-[4-(2-benzo[b]furanylmethyl)-2 (S)-(tert-butylcarbamoyl)piperazin-1-yl]pentanamide)+indinavir sulfate), JE-2147([2(S)-oxo-4-phenylmethyl-3(S)-[(2-methyl-3-oxy)phenylcarbonylamino]-1-oxabutyl]-4-[(2-methylphenyl)methylamino]carbonyl-4(R)-5,5-dimethyl-1,3-thiazole), BMS-232632((3S,8S,9B,12S)-3,12-bis(1,1-dimethylethyl)-8-hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[[4-(2-pyridinyl)phenyl]methyl]-2,5,6,10,13-pentaazatetradecanedicarboxylicacid dimethyl ester). DMP-850((4R,5S,6S,7R)-1-(3-amino-1H-indazol-5-ylmethyl)-4,7-dibenzyl-3-butyl-5,6-dihydroxyperhydro-1,3-diazepin-2-one),DMP-851, RO-0334649, Nar-DG-35, R-944, VX-385, TMC-114, Tipranavir,Fosamprenavir sodium, Fosamprenavir calcium, Darunavir, GW-0385, R-944,RO-033-4649, AG-1859, and the like.

The HIV integrase inhibitor is exemplified by 5-1360, L-870810, and thelike, the DNA polymerase inhibitor or DNA synthesis inhibitor isexemplified by Foscavir®, ACH-126443(L-2′,3′-didehydro-dideoxy-5-fluorocytidine), entecavir((1S,3S,4S)-9-[4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]guanine),calanolide A([10R-(10α,11β,12α)]-11,12-dihydro-12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H,10H-benzo[1,2-b:3,4-b′:5,6-b″]tripyran-2-one), calanolide B, NSC-674447 (1,1′-azobisformamide),Iscador (viscum alubm extract), Rubitecan, and the like, the HIVantisense drug is exemplified by HGTV-43, GEM-92, and the like, theanti-HIV antibody or other antibody is exemplified by NM-01, PRO-367,ID-247, Cytolin®, TNX-355 (CD4 antibody), AGT-1, PRO-140 (CCR5antibody), Anti-CTLA-4Mab, and the like, the HIV vaccine or othervaccine is exemplified by ALVAC®, AIDSVAX®, Remune®, HIVgp41 vaccine,HIVgp120 vaccine, HIVgp140 vaccine, HIVgp160 vaccine, HIVp17 vaccine,HIVp24 vaccine, HIVp55 vaccine, AlphaVax Vector System, canarypox gp160vaccine, AntiTat, MVA-F6 Nef vaccine, HIVrev vaccine, C4-V3 peptide,p2249f, VIR-201, HGP-30W, TBC-3B, PARTICLE-3B, and the like, Antiferon(interferon-α vaccine), and the like, the interferon or interferonagonist is exemplified by Sumiferon®, MultiFeron®, interferon-t,Reticulose, human leukocyte interferon 0, and the like, the CCR5antagonist is exemplified by SCH-351125 and the like, the pharmaceuticalagent acting on HIV p24 is exemplified by GPG-NH2(glycyl-prolyl-glycinamide) and the like, the HIV fusion inhibitor isexemplified by FP-21399(1,4-bis[3-[(2,4-dichlorophenyl)carbonylamino]-2-oxo-5,8-disodiumsulfonyl]naphthyl-2,5-dimethoxyphenyl-1,4-dihydrazone), T-1249,Synthetic Polymeric Construction No 3, pentafuside, FP-21399, PRO-542,Enfuvirtide, and the like, the IL-2 agonist or antagonist is exemplifiedby interleukin-2, Imunace®, Proleukin®, Multikine®, Ontak®, and thelike, the TNF-α antagonist is exemplified by Thalomid® (thalidomide),Remicade® (infliximab), curdlan sulfate, the α-glucosidase inhibitor isexemplified by Bucast® and the like, the purine nucleoside phosphorylaseinhibitor is exemplified by peldesine(2-amino-4-oxo-3H,5H-7-[(3-pyridyl)methyl]pyrrolo[3,2-d]pyrimidine) andthe like, the apoptosis agonist or inhibitor is exemplified by Arkin Z®,Panavir®, Coenzyme Q10 (2-deca(3-methyl-2-butenylene)-5,6-dimethoxy-3-methyl-p-benzoquinone), and thelike, the cholinesterase inhibitor is exemplified by Cognex® and thelike, and the immunomodulator is exemplified by Imunox®, Prokine®,Met-enkephalin(6-de-L-arginine-7-de-L-arginine-8-de-L-valinamide-adrenorphin), WF-10(10-fold dilute tetrachlorodecaoxide solution), Perthon, PRO-542, SCH-D,UK-427857, AMD-070, AK-602, and the like.

In addition, Neurotropin®, Lidakol®, Ancer 20®, Ampligen®, Anticort®,Inactivin®, and the like, PRO-2000, Rev M10 gene, HIV specific cytotoxicT cell (CTL immunotherapy, ACTG protocol 080 therapy, CD4-ζ genetherapy), SCA binding protein, RBC-CD4 complex, Motexafin gadolinium,GEM-92, CNI-1493, (t)-FTC, Ushercell, D2S, BufferGel®, VivaGel®,Glyminox vaginal gel, sodium lauryl sulfate, 2F5,2F5/2G12, VRX-496,Ad5gag2, BG-777, IGIV-C, BILR-255, and the like are exemplified.

As the “other anti-HIV activity substance” to be used for the anti-HIVcomposition of the present invention in the multiple drug combinationtherapy, preferred are an HIV reverse transcriptase inhibitor and an HIVprotease inhibitor. Two or three, or even a greater number ofpharmaceutical agents can be used in combination, wherein a combinationof pharmaceutical agents having different action mechanisms is one ofthe preferable embodiments. In addition, selection of pharmaceuticalagents free of side effect duplication is preferable.

Specific examples of the combination of pharmaceutical so agents includea combination of a group consisting of efavirenz, tenofavir,entricitabine, indinavir, nelfinavir, atazanavir, ritonavir+indinavir,ritonavir+lopinavir and ritonavir+saquinavir, didanosine+lamivudine,zidovudine+didanosine, stavudine+didanosine, zidovudine+lamivudine,stavudine+lamivudine and emtriva, and the crystal or mixed crystal ofthe present invention (Guidelines for the Use of Antiretroviral Agentsin HIV-Infected Adults and Adolescents. Aug. 13, 2001). Particularlypreferred is a combined use of two agents of the crystal or mixedcrystal of the present invention with efavirenz, indinavir, nelfinavir,tenofovir, emtricitabine, zidovudine or lamivudine, and a combined useof three agents of the crystal or mixed crystal of the present inventionwith zidovudine+lamivudine, tenofovir+lamivudine, tenofovir+zidovudine,tenofovir+efavirenz, tenofovir+nelfinavir, tenofovir+indinavir,tenofovir+emtricitabine, emtricitabine+lamivudine,emtricitabine+zidovudine, emtricitabine+efavirenz,emtricitabine+nelfinavir, emtricitabine+indinavir,nelfinavir+lamivudine, nelfinavir+zidovudine, nelfinavir+efavirenz,nelfinavir+indinavir, efavirenz+lamivudine, efavirenz+zidovudine orefavirenz+indinavir.

The production method of the crystal or mixed crystal of compound A ofthe present invention is not particularly limited, and the crystal canbe produced by a method known or methods shown in the followingExamples, and the like.

EXAMPLES

While the production method of the crystal of compound A of the presentinvention is explained in the following by referring to Examples, whichare mere examples and do not limit the present invention in any way.

Reference Example 1: Production of Crystal Form I of the Compound A

Step 1

2,4-Difluorobenzoic acid (50 g, 316 mmol) was dissolved in concentratedsulfuric acid (200 ml), and N-iodosuccinimide (68 g, 300 mmol) was addedby portions at not more than 5° C. After the completion of the addition,the mixture was stirred at the same temperature for 4.5 hr. The reactionmixture was poured into ice water (ca. 600 ml), then 10% aqueous sodiumsulfite solution was added, and the mixture was stirred. Theprecipitated solid was collected by filtration, washed with water, andvacuum dried to give crude crystals (85 g). The crude crystals obtainedin the same manner were combined (total amount 205 g), andrecrystallized from 50% aqueous ethanol (820 ml) to give2,4-difluoro-5-iodobenzoic acid (149 g, yield 73%) as a white solid.

¹H NMR (CDCl₃ 300 MHz) (σ) ppm: 6.94 (1H, dd, J=10.3, 10.3 Hz), 8.46(1H, d, J=7.5 Hz)

Step 2

The compound (148 g, 521 mmol) obtained in Step 1 was dissolved intoluene (750 ml), thionyl chloride (76 ml, 1.04 mol) anddimethylformamide (catalytic amount) were added, and the mixture washeated under reflux for 2 hr. The insoluble material was filtered off at60° C., and the filtrate was concentrated under reduced pressure andazeotroped with toluene (330 ml). The residue was dissolved intetrahydrofuran (400 ml), and this solution was added dropwise to asolution of ethyl 3,3-dimethylaminoacrylate (82 g, 573 mmol) andtriethylamine (87 ml, 625 mmol) in tetrahydrofuran (400 ml), and themixture was heated under reflux for 7 hr. The reaction mixture wasallowed to cool to room temperature, and concentrated under reducedpressure. Water (700 ml) and ethyl acetate (800 ml) were added forpartitioning. The organic layer was washed successively with saturatedaqueous sodium hydrogen carbonate (250 ml, X2), water (300 ml) andsaturated brine (300 ml), and dried over sodium sulfate. Afterfiltration of insoluble material, the filtrate was concentrated underreduced pressure to give a crude product (210 g) of2-(2,4-difluoro-5-iodobenzoyl)-3-dimethylaminoacrylic acid ethyl esteras a brown solid.

Step 3

The crude product (210 g) obtained in Step 2 was dissolved intetrahydrofuran (500 ml), (S)-(+)-valinol (54 g, 521 mmol) was added,and the mixture was stirred at room temperature for 30 min. The reactionmixture was concentrated under reduced pressure, and the residue wasdissolved in dimethylformamide (600 ml). Potassium carbonate (144 g,1.04 mol) was added and the mixture was stirred with heating at 70° C.for 2 hr. The reaction mixture was allowed to cool, and added to water(1500 ml) and stirred. The precipitated solid was collected byfiltration and the obtained solid was washed successively with 30%aqueous ethanol (500 ml) and a mixed solvent of diethyl ether (150 ml)and hexane (150 ml) and vacuum dried to give7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (179 g, yield 760) as a beige solid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.6 Hz), 1.10 (3H, d,J=6.6 Hz), 1.28 (3H, t, J=7.0 Hz), 2.27 (1H, br), 3.77 (1H, br), 3.86(1H, br), 4.23 (2H, q, J=7.0 Hz), 4.56 (1H, br), 5.12 (1H, t, J=4.9 Hz),8.09 (1H, d, J=11.1 Hz), 9.62 (1H, d, J=7.5 Hz), 8.68 (1H, s)

MS(ESI): M+ 448

Step 4

wherein TBDMS means a tert-butyldimethylsilyl group.

The compound (80 g, 179 mmol) obtained in Step 3 was dissolved indimethylformamide (320 ml), imidazole (16 g, 233 mmol) andtert-butyldimethylsilyl chloride (30 g, 197 mmol) were added, and themixture was stirred at room temperature for 1.5 hr. Water was added tothe reaction mixture and the mixture was extracted with ethyl acetate.The organic layer was washed successively with saturated aqueousammonium chloride solution and saturated brine, and dried over sodiumsulfate. The organic layer was filtered and the filtrate wasconcentrated under reduced pressure. The obtained residue was purifiedby silica gel chromatography (ethyl acetate:hexane-1:3 to 1:2) to give1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (77 g, yield 77%) as a colorless amorphous form.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.07 (3H, s), −0.05 (3H, s), 0.77 (9H,s), 0.84 (3H, d, J=6.5 Hz), 1.18 (3H, d, J=6.5 Hz), 1.40 (3H, t, J=7.2Hz), 2.35-2.50 (1H, m), 3.85-3.95 (1H, m), 3.98-4.10 (2H, m), 4.30-4.40(2H, m), 7.26 (1H, s), 8.64 (1H, s), 8.94 (1H, d, J=7.2 Hz)

MS (ESI): M+ 562

Step 5

Preparation of a Solution of 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran

Under an argon stream, zinc powder (11 g, 267 mmol) was suspended intetrahydrofuran (30 ml), 1,2-dibromoethane (0.15 ml, 1.8 mmol) andtrimethylsilyl chloride (0.45 ml, 3.6 mmol) were added at 65° C., andthe mixture was stirred with heating for 30 min. A solution of3-chloro-2-fluorobenzyl bromide (41 g, 178 mmol) in tetrahydrofuran (100ml) was added dropwise at 65° C., and the mixture was stirred withheating for 2 hr and allowed to cool to room temperature to give asolution of 1M 3-chloro-2-fluorobenzylzinc bromide in tetrahydrofuran.This was used in the next main step.

Main Step

The compound (76 g, 136 mmol) obtained in Step 4 was dissolved intetrahydrofuran (600 ml) and, under an argon stream,dibenzylidenacetonepalladium(II) (3.2 g, 5.5 mmol) and trifurylphosphine(2.6 g, 11.0 mmol) were added, and a solution of the aforementioned 1M3-chloro-2-fluorobenzylzinc bromide in tetrahydrofuran (178 ml, 178mmol) was added dropwise at 60° C. After the completion of the dropwiseaddition, the mixture was stirred with heating at the same temperaturefor 2 hr. The reaction mixture was allowed to cool to room temperature,saturated aqueous ammonium chloride solution was added, and the mixturewas filtered through celite. The filtrate was extracted twice with ethylacetate. The organic layer was washed successively with water (twice)and saturated brine, and dried over magnesium sulfate. The organic layerwas filtered and the filtrate was concentrated under reduced pressure,and the obtained residue was purified by silica gel chromatography(chloroform:acetone=40:1) to give1-((S)-1-tert-butyldimethyluilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (68 g, yield 84%) as a colorless amorphous form.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.09 (3H, s), −0.05 (3H, s), 0.75 (9H,s), 0.85 (3H, d, J=6.7 Hz), 1.18 (3H, d, 6.7 Hz), 1.39 (3H, t, J=7.1Hz), 2.45 (1H, br), 3.89-3.92 (1H, m), 3.98-4.02 (1H, m), 4.07-4.12 (1H,m), 4.12 (2H, s), 4.34-4.41 (2H, m), 6.96-7.00 (1H, m), 7.03-7.05 (1H,m), 7.21-7.24 (1H, m), 7.26-7.29 (1H, m), 8.39 (1H, d, J=8.8 Hz), 8.63(1H, s)

Step 6

The compound (48 g, 86 mmol) obtained in Step 5 was dissolved inmethanol (300 ml), water (5 ml) and 28% sodium methoxide methanolsolution (176 ml, 862 mmol) were added, and the mixture was heated underreflux for 24 hr. The reaction mixture was allowed to cool to roomtemperature and the mixture was neutralized by adding 6N hydrochloricacid. Methanol was evaporated under reduced pressure. Water was added tothe obtained solution and the mixture was stirred. The precipitatedsolid was collected by filtration and the obtained solid was dissolvedin ethyl acetate. The mixture was washed with water and dried oversodium sulfate. The solution was filtered and the filtrate wasconcentrated under reduced pressure. The obtained residue wasrecrystallized from ethyl acetate-hexane to give a compound (32 g, yield86%) as a white solid. The obtained compound (32 g) was dissolved inbutyl acetate (160 ml) by heating under reflux, and crystal form II wasseeded at 75° C. s. The mixture was stirred for 3.5 hr while allowing tocool as it was. The precipitated solid was collected by filtration,washed with butyl acetate (25 ml) and vacuum dried to give a compound(25 g, yield 77%) as a white solid. The obtained compound (4.0 g) wasdissolved in methanol (40 ml) by heating under reflux at 50° C., andadded dropwise to water (40 ml) at room temperature. The mixture wasstirred at room temperature for 16 hr, filtered, and the remaining solidwas washed with 66% aqueous methanol, and vacuum dried to give a crystalof compound A (crystal form I) (3.9 g, yield 97%) as a white solid.

m.p. 151-152° C.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1H, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1H, s), 8.88 (1H, s), 15.44 (1H, s)

MS (ESI): M+ 446

Example 1: Production of Crystal Form II of the Compound A

Step 1

2,4-Difluorobenzoic acid (50 g, 0.316 mmol) was dissolved inconcentrated sulfuric acid (200 ml), and N-iodosuccinimide (68 g, 300mmol) was added by portions at not more than 5° C. After the completionof the addition, the mixture was stirred at the same temperature for 4.5hr. The reaction mixture was poured into ice water (ca. 600 ml), then10% aqueous sodium sulfite solution was added, and the mixture wasstirred. The precipitated solid was collected by filtration, washed withwater, and vacuum dried to give crude crystals (85 g). The crudecrystals obtained in the same manner were combined (total amount 205 g),and recrystallized from 50% aqueous ethanol (820 ml) to give2,4-difluoro-5-iodobenzoic acid (148 g, yield 73%) as a white solid.

¹H NMR (CDCl₃ 300 MHz) (σ) ppm: 6.94 (1H, dd, J=10.3, 10.3 Hz), 8.46(1H, d, J=7.5 Hz)

Step 2

The compound (148 g, 531 mmol) obtained in Step 1 was dissolved intoluene (750 ml), thionyl chloride (76 ml, 1.04 mol) anddimethylformamide (catalytic amount) were added, and the mixture washeated under reflux for 2 hr. The insoluble material was filtered off at60° C., and the filtrate was concentrated under reduced pressure andazeoptoped with toluene (330 ml). The residue was dissolved intetrahydrofuran (400 ml), and this solution was added dropwise to asolution of ethyl 3,3-dimethylaminoacrylate (82 g, 573 mmol) andtriethylamine (87 ml, 625 mmol) in tetrahydrofuran (400 ml), and themixture was heated under reflux for 7 hr. The reaction mixture wasallowed to cool to room temperature and concentrated under reducedpressure. Water (700 ml) and ethyl acetate (800 ml) were added to allowpartitioning. The organic layer was washed successively with saturatedaqueous sodium hydrogen carbonate (250 ml) twice, water (300 ml) andsaturated brine (300 ml), and dried over sodium sulfate. The mixture wasfiltered, and the filtrate was concentrated under reduced pressure togive a crude product (210 g) of2-(2,4-difluoro-5-iodobenzoyl)-3-dimethylaminoacrylic acid ethyl esteras a brown solid.

Step 3

The crude product (210 g) obtained in Step 2 was dissolved intetrahydrofuran (500 ml), (S)-(+)-valinol (54 g, 0.521 mmol) was added,and the mixture was stirred at room temperature for so 30 min. Thereaction mixture was concentrated under reduced pressure, and theresidue was dissolved in dimethylformamide (600 ml). Potassium carbonate(144 g, 1.04 mol) was added, and the mixture was stirred with heating at70° C. for 2 hr. The reaction mixture was allowed to cool to roomtemperature, added to water (1500 ml) and the mixture was stirred. Theprecipitated solid was collected by filtration. The obtained solid waswashed successively with 30% aqueous ethanol (500 ml) and a mixedsolvent of diethyl ether (150 ml) and hexane (150 ml), and vacuum driedto give7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (178 g, yield 76% (relative to Step 2)) as a beigesolid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.6 Hz), 1.10 (3H, d,J=6.6 Hz), 1.28 (3H, t, J=7.0 Hz), 2.27 (1H, br), 3.77 (1H, br), 3.86(1H, br), 4.23 (2H, q, J=7.0 Hz), 4.56 (1H, br), 5.12 (1H, t, J=4.9 Hz),8.09 (1H, d, J=11.1 Hz), 8.62 (1H, d, J=7.5 Hz), 8.68 (1H, s)

MS (ESI): M+ 448

Step 4

The compound (150 g, 335 mmol) obtained in Step 3 was dissolved indimethylformamide (500 ml), imidazole (30 g, 0.436 mmol) andtert-butyldimethylsilyl chloride (56 g, 369 mmol) were added, and themixture was stirred at room temperature for 1.5 hr. Water was added tothe reaction mixture and extracted with ethyl acetate. The organic layerwas washed successively with water, saturated aqueous ammonium chloridesolution and saturated brine, and dried over sodium sulfate. The organiclayer was filtered and the filtrate was concentrated under reducedpressure, and the obtained residue was purified by silica gelchromatography (ethyl acetate:hexane-1:3 to 1:2) to give1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (173 g, yield 92%) as a colorless amorphous form.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.07 (3H, s), −0.05 (3H, s), 0.77 (9H,s), 0.84 (3H, d, J=6.5 Hz), 1.18 (3H, d, J=6.5 Hz), 1.40 (3H, t, J=7.2Hz), 2.35-2.50 (1H, m), 3.85-3.95 (1H, m), 3.98-4.10 (2H, m), 4.30-4.40(2H, m)), 7.26 (1H, s), 8.64 (1H, s), 8.94 (1H, d, J=7.2 Hz)

MS (ESI): M+ 562

Step 5

Preparation of a Solution of 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran

Under an argon stream, zinc powder (11 g, 175 mmol) was suspended intetrahydrofuran (30 ml), 1,2-dibromoethane (0.1 ml, 1.20 mmol) andtrimethylsilyl chloride (0.29 ml, 2.4 mmol) were added at 60° C., andthe mixture was stirred with heating for 30 min. A solution of3-chloro-2-fluorobenzyl bromide (27 g, 119 mmol) in tetrahydrofuran (60ml) was added dropwise at 60° C. The mixture was stirred with heatingfor 1 hr and allowed to cool to room temperature to give a solution of1M 3-chloro-2-fluorobenzylzinc bromide in tetrahydrofuran. This was usedin the next main step.

Main Step

The compound (50 g, 89 mmol) obtained in Step 4 was dissolved intetrahydrofuran (400 ml) and, under an argon stream,dichlorobis(triphenylphosphine)palladium(II) (2.1 g, 3.6 mmol) was addedand a solution of the above-mentioned 1M 3-chloro-2-fluorobenzylzincbromide in tetrahydrofuran was added dropwise at 60° C. After thecompletion of the dropwise addition, the mixture was stirred withheating at the same temperature for 1.5 hr. The reaction mixture wasallowed to cool to room temperature, IN hydrochloric acid was added andthe mixture was extracted 3 times with ethyl acetate. The organic layerwas washed successively with water and saturated brine, and dried overmagnesium sulfate. The organic layer was filtered and the filtrate wasconcentrated under reduced pressure, and the obtained residue waspurified by silica gel chromatography (ethyl acetate:hexane-1:2 to 1:1)to give1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (43 g, yield 83%) as a brown amorphous form.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.09 (3H, s), −0.05 (3H, s), 0.75 (9H,s), 0.85 (3H, d, J=6.7 Hz), 1.18 (3H, d, 6.7 Hz), 1.39 (3H, t, J=7.1Hz), 2.45 (1H, br), 3.89-3.92 (1H, m), 3.98-4.02 (1H, m), 4.07-4.12 (1K,m), 4.12 (2H, s), 4.34-4.41 (2H, m), 6.96-7.00 (1H, m), 7.03-7.05 (1H,m), 7.21-7.24 (1H, m), 7.26-7.29 (1H, m), 6.39 (1H, d, J=8.8 Hz), 8.63(1H, s)

Step 6

The compound (43 g, 74 mmol) obtained in Step 5 was dissolved inmethanol (280 ml), 28% sodium methoxide methanol solution (151 ml, 742mmol) and water (4.3 ml) were added, and the mixture was heated underreflux for 20 hr. The reaction mixture was filtered through celite. Thefiltrate was concentrated under reduced pressure. Water (400 ml) wasadded to the residue, and the mixture was washed with hexane (100 ml).The aqueous layer was acidified by adding concentrated hydrochloric acid(65 ml), and the mixture was extracted with ethyl acetate. The organiclayer was washed successively with water and saturated brine, and driedover sodium sulfate. The solution was filtered and the filtrate wasconcentrated under reduced pressure. The obtained crude product (35 g,brown oil) was dissolved in ethyl acetate (49 ml) by heating underreflux, hexane (30 ml) was added while allowing to cool, and the mixturewas stirred for 18.5 hr. The precipitated solid was collected byfiltration, washed with a mixed solvent of ethyl acetate and hexane(1:1), and vacuum dried to give a crystal of compound A (crystal formII) (27 g, yield 82%) as a white solid.

m.p. 153.7-153.9° C.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1H, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1H, s), 8.88 (1H, s), 15.44 (1H, s)

MS (ESI): M+ 448

Example 2: Production of Crystal Form II of the Compound A Example 2-1:Production of Crystal Form II of the Compound A

Step 1

2,4-Difluorobenzoic acid (100 g, 633 mmol) was dissolved in concentratedsulfuric acid (400 ml), and N-iodosuccinimide (142 g, 601 mol) was addedby portions at not more than 5° C. After the completion of the addition,the mixture was stirred at the same temperature for 6 hr. The reactionmixture was poured into ice water (ca. 2400 ml), then saturated aqueoussodium sulfite solution was added, and the mixture was stirred. Theprecipitated solid was collected by filtration, washed with water, andvacuum dried to give crude crystals (188 g). The crude crystals obtainedin the same manner were combined (total amount 568 g), andrecrystallized from 50% aqueous ethanol (2600 ml) to give2,4-difluoro-5-iodobenzoic acid (388 g, yield 68%) as a white solid.

¹H NMR (CDCl₃ 300 MHz) (σ) ppm: 6.94 (1H, dd, J=10.3, 10.3 Hz), 8.46(1H, d, J=7.5 Hz)

Step 2

The compound (200 g, 704 mmol) obtained in Step 1 was dissolved intoluene (1000 ml), thionyl chloride (103 ml, 408 mmol) anddimethylformamide (catalytic amount) were added, and the mixture washeated under reflux for 2 hr. The insoluble material was filtered off,and the filtrate was concentrated under reduced pressure and azeotropedwith toluene. The residue was dissolved in tetrahydrofuran (500 ml),this solution was added dropwise to a solution of ethyl3,3-dimethylaminoacrylate (111 g, 775 mmol) and triethylamine (118 ml,845 mmol) in tetrahydrofuran (500 ml), and the mixture was heated underreflux for 3 hr. The reaction mixture was allowed to cool to roomtemperature and filtered and the filtrate was concentrated under reducedpressure. Water (500 ml) and ethyl acetate (800 ml) were added to allowpartitioning. The organic layer was washed successively with saturatedaqueous sodium hydrogen carbonate (200 ml), water (200 ml) and saturatedbrine, and dried over sodium sulfate. The organic layer was filtered,and the filtrate was concentrated under reduced pressure to give a crudeproduct (273 g) of 2-(2,4-difluoro-5-iodobenzoyl)-3-dimethylaminoacrylicacid ethyl eater as a brown solid.

Step 3

The crude product (273 g) obtained in Step 2 was dissolved intetrahydrofuran (650 ml), (S)-(+)-valinol (73 g, 708 mmol) was added,and the mixture was stirred at room temperature for 2 hr. The reactionmixture was concentrated under reduced pressure and the residue wasdissolved in dimethylformamide (800 ml). Potassium carbonate (195 g 1.41mol) was added, and the mixture was stirred with heating at 70° C. for2.5 hr. The reaction mixture was allowed to cool to room temperature,added to water (2000 ml) and the mixture was stirred. The precipitatedsolid was collected by filtration. The obtained solid was subject toslurry washing successively with water and 301 aqueous ethanol (650 ml)and vacuum dried to give a crude product (217 g). The obtained crudeproduct (217 g) was subject to slurry washing with a mixed solvent ofethyl acetate (650 ml) and hexane (440 ml) with heating under reflux.The mixture was filtered, and the remaining solid was vacuum dried togive7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (207 g, yield 66% (relative to Step 2)) as a pale-brownsolid.

¹H NM (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.6 Hz), 1.10 (3H, d,J=6.6 Hz), 1.28 (3H, t, J=7.0 Hz), 2.27 (1H, br), 3.77 (1H, br), 3.86(1H, br), 4.23 (2H, q, J=7.0 Hz), 4.56 (1H, br), 5.12 (1H, t, J=4.9 Hz),8.09 (1H, d, J=11.12 Hz), 8.62 (1H, d, J=7.5 Hz), 8.68 (1H, s)

MS(ESI): M+ 448

Step 4

The compound (150 g, 335 mmol) obtained in Step 3 was dissolved indimethylformamide (450 ml), imidazole (27 g, 397 mmol) andtert-butyldimethylsilyl chloride (58 g, 385 mmol) were added, and themixture was stirred overnight at room temperature. Water (900 ml) wasadded to the reaction mixture, and the mixture was extracted with ethylacetate (680 ml). The organic layer was washed successively with water(450 ml, 3 times) and 2a saturated brine (200 ml), and dried over sodiumsulfate. The organic layer was filtered, and the filtrate wasconcentrated under reduced pressure to give a crude product (192 g) of1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester as a pale-yellow amorphous form.

Step 5

The crude product (162 g) obtained in Step 4 was dissolved intetrahydrofuran (160 ml) and, under an argon stream, dibenzylidenacetonepalladium(II) (1.7 g, 2.9 mmol) and trifurylphosphine (1.3 g, 5.8 mmol)were added. To this mixture was added dropwise at 60° C. a solution of(375 ml, 375 mmol) of IM 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran obtained in the same manner as in Example 1, Step 5 and,after the completion of the dropwise addition, the mixture was stirredwith heating at the same temperature for 3.5 hr. The reaction mixturewas allowed to cool to room temperature, ethyl acetate (640 ml) and 10%aqueous citric acid solution (400 ml) were added, and the mixture wasfiltered through Celite, and the filtrate was partitioned. The organiclayer was washed successively with water (200 ml), saturated aqueoussodium hydrogen carbonate (400 ml) and saturated brine (200 ml), anddried over sodium sulfate. The organic layer was filtered, and thefiltrate was concentrated under reduced pressure to give a crude product(186 g) of1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester as a brown oil.

Step 6

The crude product (193 g) obtained in Step 5 was dissolved inisopropanol (650 ml), IN aqueous sodium hydroxide solution (1290 ml,1.29 mol) was added, and the mixture was heated under reflux for 2 hr.The reaction mixture was allowed to cool to room temperature, andfiltered through Celite. The filtrate was acidified by addingconcentrated hydrochloric acid and the mixture was stirred. Theprecipitated solid was collected by filtration, and vacuum dried to givea crude product (132 g) as a pale-yellow solid. The crude productsobtained in the same manner were combined (total amount 143 g),suspended in butyl acetate (430 ml) and subject to slurry stirring withheating under reflux for 1 hr. The suspension was allowed to cool toroom temperature and filtered and vacuum dried to give6-(3-chloro-2-fluorobenzyl)-7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (99 g, yield 74% (relative to Step 3)) as a gray solid.

¹H NMR (DMSO-d₆ 400 MHz) (δ) ppm: 0.71 (3H, d, J=6.5 Hz), 1.13 (3H, d,J=6.5 Hz), 2.36 (1H, br), 3.77 (1H, br), 3.94 (1H, br), 4.25 (2H, m),4.77 (1H, br), 5.16 (1H,

t, J=2.4 Hz), 7.19-7.23 (1H, m), 7.32-7.35 (1H, m), 7.48-7.52 (1H, m),8.24-8.28 (2H, m), 9.00 (1H, s), 15.00 (1H, s)

MS (ESI): M+ 436

Step 7

The compound (99 g, 227 mmmol) obtained in Step 6 was dissolved inmethanol (530 ml), 28% sodium methoxide methanol solution (465 ml, 2.28mol) was added, and the mixture was heated under reflux for 20 hr. Thereaction mixture was allowed to cool to room temperature and filteredthrough Celite. The filtrate was concentrated under reduced pressure.The residue was acidified by adding water (200 ml) and concentratedhydrochloric acid (190 ml), and extracted with ethyl acetate (500 ml).The organic layer was washed twice with water (200 ml), and dried oversodium sulfate. The mixture was filtered, and the filtrate wasconcentrated under reduced pressure to give a crude product (108 g). Theobtained crude product (108 g) was dissolved in isobutyl acetate (330ml) with heating and the mixture was stirred while allowing to cool for24 hr. The precipitated solid was collected by filtration, and vacuumdried to give compound A (71 g, yield 69%) as a white solid. The crudecrystals obtained in the same manner were combined (total amount 233 g),dissolved in isobutyl acetate (470 ml) by heating under reflux, and themixture was stirred overnight while allowing to cool. The precipitatedsolid was collected by filtration, and vacuum dried to give a crystal ofcompound A (crystal form II) (206 g, yield 88%) as a white solid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1N, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1N, a), 8.88 (1N, s), 15.44 (1H, s)

MS (ESI): M+ 448

Example 2-2: Production of Crystal Form II of the Compound A

Step 1

5-Bromo-2,4-difluorobenzoic acid (82.7 kg, 349 mol) was dissolved intoluene (420 L), thionyl chloride (62.3 kg, 523 mol) anddimethylformamide (catalytic amount) were added, and the mixture wasstirred at 70° C. for 6 hr. The reaction mixture was allowed to cool toroom temperature, concentrated under reduced pressure, and azeotropedagain with toluene (420 L). The residue was dissolved in toluene (220L), this solution was added dropwise to a solution of ethyl3,3-dimethylaminoacrylate (55.0 kg, 384 mol) and diisopropylethylamine(58.6 kg, 523 mol) in toluene (220 L), and the mixture was stirred withheating at 70° C. for 21 hr. The reaction mixture was allowed to cool toroom temperature, (S)-(+)-valinol (36.0 kg, 349 mol) was added, and themixture was stirred at room temperature for 1.5 hr. Water (420 L) wasadded to the reaction mixture to allow partitioning, and the organiclayer was washed successively with 1N hydrochloric acid (250 L, twice),water (420 L), 5% aqueous sodium hydrogen carbonate (250 L, twice),water (420 L) and 10% brine (250 L). The extract was concentrated underreduced pressure and azeotroped with dimethylformamide (420 L) to give aconcentration residue (330 L) containing a crude product of2-(5-bromo-2,4-difluorobenzoyl)-3-((S)-1-hydroxymethyl-2-methylpropylmethylamino)acrylicacid ethyl ester.

Step 2

To a solution (330 L) of the crude product obtained in Step 1 indimethylformamide was added 1,8-diazabicyclo[5.4.0]undecane (105 kg, 349mol) and the mixture was stirred at room temperature for 23 hr. To thereaction mixture were added dimethylformamide (330 L), and then water(170 L) and, after stirring for 2 hr, water (170 L) was added dropwise.The precipitated solid was collected by filtration and washed withdimethylformamide (170 L)-water (170 L) mixture, and then with ethanol(460 L)-water (200 L) mixed solution. The obtained solid was vacuumdried, suspended in an ethyl acetate (330 L)-n-heptane (330 L) mixtureand subject to slurry washing. The suspension was filtered, and theremaining solid was vacuum dried to give6-bromo-7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (102 kg, yield 73%) as a yellow white solid. Thiscompound was confirmed to be equivalent to the standard product of thecompound by high performance liquid chromatography (HPLC) analysis.

Step 3

The compound (45.0 kg, 112 mol) obtained in Step 2 and imidazole (9.95kg, 146 mol) were suspended in toluene (180 L), a solution oftert-butyldimethylsilyl chloride (17.8 kg, 118 mol) in toluene (45 L)was added at 50° C., and the mixture was stirred at the same temperaturefor 3 hr. Toluene (230 L) was added to the reaction mixture, and washedsuccessively with water (450 L, twice) and 20% brine (450 L). Theextract was concentrated under reduced pressure and azeotroped withtetrahydrofuran (320 L) to give a concentration residue (390 L)containing a crude product of6-bromo-1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester.

Step 4

Preparation of a Solution of 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran

Under a nitrogen stream, zinc powder (18.8 kg, 287 mol) was suspended intetrahydrofuran (130 L), 1,2-dibromoethane (470 g, 2.50 mol) was addedat 60° C., and the mixture was stirred at the same temperature for 30min. Trimethylsilyl chloride (560 g, 3.10 mol) was added to thissuspension at room temperature, and the mixture was stirred with heatingfor 30 min. A solution of 3-chloro-2-fluorobenzyl bromide (54.0 kg, 242mol) in tetrahydrofuran (65 L) was added dropwise at 0° C., and themixture was stirred at 20° C. for 3 hr. The remaining zinc was filteredoff to give a solution of 1M 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran. This was used in the next main step.

Main Step

Under a nitrogen stream, tris(dibenzylidenacetone)dipalladium(0) (1.96kg, 3.36 mol) and triphenylphosphine (1.77 kg, 6.72 mol) were dissolvedin tetrahydrofuran (180 L), and the mixture was stirred at roomtemperature for 1 hr. A solution (390 L) of the crude product obtainedin Step 3 in tetrahydrofuran was added dropwise at room temperature andwashed with tetrahydrofuran (45 L). A solution (164 kg, 157 mol) of theabove-mentioned IN 3-chloro-2-fluorobenzylzinc bromide intetrahydrofuran prepared in advance was added dropwise at roomtemperature, and the mixture was stirred with heating at 55° C. for 5hr. The reaction mixture was allowed to cool to room temperature,toluene (230 L) and 25% aqueous ammonium chloride solution (230 L) wereadded and the mixture was stirred. After filtration, the mixture waspartitioned. The organic layer was washed successively with 25% aqueousammonium chloride solution (230 L), water (230 L), 5% aqueous sodiumhydrogen carbonate (230 L, 3 times) and 10% brine (230 L). The extractwas concentrated under reduced pressure to give a crude product (80 L)of6-(3-chloro-2-fluorobenzyl)-7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid as a brown oil.

Step 5

The crude product (80 L) obtained in Step 4 was dissolved in isopropanol(180 L), IN aqueous sodium hydroxide solution (180 L, 180 mol) wasadded, and the mixture was stirred with heating at 50° C. for 9 hr.Activated carbon (4.5 kg) was added to as the reaction mixture. Themixture was stirred at room temperature for 30 min, filtered throughcellulose powder and thoroughly washed with an isopropanol (45 L)—water(45 L) mixture. Water (180 L) and n-heptane (230 L) were added to thefiltrate and, after stirring, the mixture was partitioned. The aqueouslayer was washed again with n-heptane (230 L). 4N Hydrochloric acid (45L, 180 mol) and methyl isopropyl ketone (450 L) were added to theorganic layer and, after stirring, the mixture was partitioned. Theorganic layer was washed successively with 10% brine (230 L), twice with8.5% aqueous sodium hydrogen carbonate (230 L), 0.5N hydrochloric acid(230 L) and water (230 L). The extract was concentrated under reducedpressure, azeotroped 3 times with toluene (230 L). The residue wasstirred at 100° C. for 1.5 hr, allowed to cool to room temperature andstirred for 3 hr. The precipitated solid was collected by filtration andthe obtained solid was washed with toluene (45 L) and vacuum dried togive6-(3-chloro-2-fluorobenzyl)-7-fluoro-1-[(S)-1-hydroxymethyl-2-methylpropyl]-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (42.5 kg, yield 87%) as a pale-yellow solid. This compound wasconfirmed to be equivalent to the standard product by HPLC analysis.

Step 6

The compound (39.2 kg, 89.9 mol) obtained in Step 5 was dissolved inmethanol (240 L), 28% sodium methoxide methanol solution (173 kg, 899mol) was added dropwise at 10° C., and the mixture was stirred withheating at 70° C. for 21 hr. Activated carbon (3.9 kg) was added to thereaction mixture. The mixture was stirred at room temperature for 1 hr,filtered through cellulose powder and thoroughly washed with methanol(80 L). Water (29 kg, 1620 mol) was added to the filtrate and themixture was concentrated under reduced pressure. The residue wasazeotroped twice with isopropanol (240 L, 120 L). To the residue wereadded 15% brine (200 L) and toluene (200 L) and, after stirring, themixture was partitioned. The organic layer was washed successively with20% brine (200 L, 3 times), 0.5N hydrochloric acid (200 L) containingsodium chloride (10 kg) and 20% brine (200 L). The organic layer wasconcentrated under reduced pressure and azeotroped with ethyl acetate(200 L). Ethyl acetate (320 L) and water (200 L) were added to theresidue and, after stirring, the mixture was partitioned. The organiclayer was concentrated under reduced pressure and azeotroped twice withisobutyl acetate (200 L). The residue was dissolved by heating filteredwhile it was hot, and thoroughly washed with isobutyl acetate (20 L). Aseed crystal (crystal form II of compound A, 39 g) was added to thefiltrate at 60° C., and the mixture was stirred at the same temperaturefor 1.5 hr. The mixture was stirred with heating at 80° C. for 2 hr,allowed to cool to room temperature and further stirred for 6 hr. Theprecipitated solid was collected by filtration. The obtained solid waswashed with isobutyl acetate (40 L) and vacuum dried to give a crystalof compound A (crystal form II) (29.0 kg, yield 72%) as a white solid.This crystal was confirmed to be equivalent to the standard product ofthe crystal (crystal form II of compound A obtained in Example 2-1) byHPLC and X ray powder diffraction (XRPD) analysis.

Example 2-3: Production of Crystal Form II of the Compound A

The crystal form II can be also produced by crystallization according tothe methods described in Examples 2-3-1 to 2-3-26.

Example 2-3-1

The compound A (200 mg) obtained in Example 1 was dissolved in 1-butanol(2 ml) with heating under reflux. The mixture was stirred for 17 hrwhile allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (125 mg, yield 63%)of compound A as a white solid.

Example 2-3-2

The compound A (200 mg) obtained in Example 1 was dissolved in butylacetate (2 ml) with heating under reflux. The mixture was stirred for 17hr while allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (0.102 mg, yield51%) of compound A as a white solid.

Example 2-3-3

The compound A (200 mg) obtained in Example 1 was dissolved in methylisobutyl ketone (2 ml) with heating under reflux. Heptane (2 ml) wasadded dropwise and the mixture was stirred for 6 hr while allowing tocool. The precipitated solid was collected by filtration and vacuumdried to give crystal form II (168 mg, yield 84%) of compound A as awhite solid.

Example 2-3-4

The compound A (200 mg) obtained in Example 1 was dissolved in ethanol(2 ml) with heating under reflux. The mixture was stirred for 17 hrwhile allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (56 mg, yield 28%)of compound A as a white solid.

Example 2-3-5

The compound A (200 mg) obtained in Example 1 was dissolved in ethylacetate (2 ml) with heating under reflux. Heptane (1.6 as ml) was addeddropwise and the mixture was stirred for 6 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (166 mg, yield 83%) of compound A as a white solid.

Example 2-3-6

The compound A (200 mg) obtained in Example 1 was dissolved in methylethyl ketone (2 ml) with heating under reflux. Heptane (4 ml) was addeddropwise and the mixture was stirred for 6 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II 23 (123 mg, yield 62%) of compound A as a whitesolid.

Example 2-3-7

The compound A (200 mg) obtained in Example 1 was dissolved in1-propanol (2 ml) with heating under reflux. The mixture was stirred for17 hr while allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (91 mg, yield 46%)of compound A as a white solid.

Example 2-3-8

The compound A (200 mg) obtained in Example 1 was dissolved inisopropanol (2 ml) with heating under reflux. The mixture was stirredfor 17 hr while allowing to cool. The precipitated solid was collectedby filtration and vacuum dried to give crystal form II (88 mg, yield44%) of compound A as a white solid.

Example 2-3-9

The compound A (200 mg) obtained in Example 1 was dissolved in cumene (2ml) with heating under reflux. The mixture was stirred for 17 hr whileallowing to cool. The precipitated solid was collected by filtration andvacuum dried to give crystal form II (188 mg, yield 94%) of compound Aas a white solid.

Example 2-3-10

The compound A (200 mg) obtained in Example 1 was dissolved in anisole(2 ml) with heating under reflux. The mixture was stirred for 17 hrwhile allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (107 mg, yield 54%)of compound A as a white solid.

Example 2-3-11

The compound A (200 mg) obtained in Example 1 was dissolved in acetone(2 ml) with heating under reflux. Heptane (2 ml) was added dropwise andthe mixture was stirred for 16.5 hr while allowing to cool. Heptane (4ml) was further added, and the mixture was further stirred for 24 hr.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (134 mg, yield 67%) of compound A as a white solid.

Example 2-3-12

The compound A (200 mg) obtained in Example 1 was dissolved in ethanol(2 ml) with heating under reflux. Heptane (4 ml) was added dropwise andthe mixture was stirred for 19 hr while allowing to cool. Theprecipitated solid was collected by filtration and vacuum dried to givecrystal form II (129 mg, yield 65%) of compound A as a white solid.

Example 2-3-13

The compound A (200 mg) obtained in Example 1 was dissolved inisopropanol (2 ml) with heating under reflux. Heptane (4 ml) was addeddropwise and the mixture was stirred for 19 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (166 mg, yield 83%) of compound A as a white solid.

Example 2-3-14

The compound A (200 mg) obtained in Example 1 was dissolved in1-propanol (2 ml) with heating under reflux. Heptane (4 ml) was addeddropwise and the mixture was stirred for 19 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (158 mg, yield 79%) of compound A as a white solid.

Example 2-3-15

The compound A (200 mg) obtained in Example 1 was dissolved inisobutanol (2 ml) with heating under reflux. The mixture was stirred for21 hr while allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (131 mg, yield 66%)of compound A as a white solid.

Example 2-3-16

The compound A (200 mg) obtained in Example 1 was dissolved in toluene(2 ml) with heating at 100° C. The mixture was stirred for 37 hr whileallowing to cool. The precipitated solid was collected by filtration andvacuum dried to give crystal form II (190 mg, yield 95%) of compound Aas a white solid.

Example 2-3-17

The compound A (200 mg) obtained in Example 1 was dissolved in methylbutyl ketone (2 ml) with heating at 60° C. Heptane (1.8 ml) was addeddropwise and the mixture was stirred for 37 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (191 mg, yield 96%) of compound A as a white solid.

Example 2-3-18

The compound A (200 mg) obtained in Example 1 was dissolved inchloroform (1 ml) with heating at 60° C. Isopropyl ether (1.8 ml) wasadded dropwise and the mixture was stirred for 37 hr while allowing tocool. The precipitated solid was collected by filtration and vacuumdried to give crystal form II (184 mg, yield 92%) of compound A as awhite solid.

Example 2-3-19

The compound A (200 mg) obtained in Example 1 was dissolved intetrahydrofuran (1 ml) by heating at 60° C. Isopropyl ether (2 ml) wasadded dropwise and the mixture was stirred for 41 hr while allowing tocool. The precipitated solid was collected by filtration and vacuumdried to give crystal form II (144 mg, yield 72%) of compound A as awhite solid.

Example 2-3-20

The compound A (200 mg) obtained in Example 1 was dissolved inisobutanol (2 ml) with heating under reflux. Heptane (2 ml) as was addeddropwise and the mixture was stirred for 21 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (160 mg, yield 80%) of compound A as a white solid.

Example 2-3-21

The compound A (200 mg) obtained in Example 1 was dissolved in butanol(2 ml) with heating under reflux. Heptane (2 ml) was added dropwise andthe mixture was stirred for 21 hr while allowing to cool. Theprecipitated solid was collected by filtration and vacuum dried to givecrystal form II (152 mg, yield 76%) of compound A as a white solid.

Example 2-3-22

The compound A (200 mg) obtained in Example 1 was dissolved in isobutylacetate (2 ml) with heating under reflux. The mixture was stirred for 21hr while allowing to cool. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (140 mg, yield 70%)of compound A as a white solid.

Example 2-3-23

The compound A (200 mg) obtained in Example 1 was dissolved 2a inisobutyl acetate (2 ml) with heating under reflux. Heptane (2 ml) wasadded dropwise and the mixture was stirred for 21 hr while allowing tocool. The precipitated solid was collected by filtration and vacuumdried to give crystal form II (178 mg, yield 89%) of compound A as awhite solid.

Example 2-3-24

The compound A (200 mg) obtained in Example 1 was dissolved in butylacetate (2 ml) with heating under reflux. Heptane (1.5 ml) was addeddropwise and the mixture was stirred for 21 hr while allowing to cool.The precipitated solid was collected by filtration and vacuum dried togive crystal form II (158 mg, yield 78%) of compound A as a white solid.

Example 2-3-25

The compound A (200 mg) obtained in Example 1 was dissolved in anisole(2 ml) by heating at 110° C. Heptane (2 ml) was added dropwise and themixture was stirred for 21 hr while allowing to cool. The precipitatedsolid was collected by filtration and vacuum dried to give crystal formII (197 mg, yield 89%) of compound A as a white solid.

Example 2-3-26

The compound A (200 mg) obtained in Example 1 was dissolved in butylacetate (2 ml) with heating under reflux. After rapid cooling, themixture was stirred for 2 hr. The precipitated solid was collected byfiltration and vacuum dried to give crystal form II (131 mg, yield 66%)of compound A as a white solid.

Example 2-4: Production of Crystal Form II of the Compound A

Step 1

1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (48 g, 86 mmol) obtained in Example 1, Step 5 wasdissolved in methanol (300 ml), water (5 ml) and 28% sodium methoxidemethanol solution (176 ml, 862 mmol) were added, and the mixture washeated under reflux for 24 hr. The reaction mixture was allowed to coolto room temperature, neutralized with 6N hydrochloric acid and methanolwas evaporated under reduced pressure. Water was added to the obtainedsolution and, after stirring, the precipitated solid was collected byfiltration. The obtained solid was dissolved in ethyl acetate, washedwith water, and dried over sodium sulfate. The mixture was filtered andthe filtrate was concentrated under reduced pressure. The obtainedresidue was recrystallized from ethyl acetate-hexane to give compound A(primary crystal 29.5 g, secondary crystal 2.8 g, in total 32.3 g, yield86%) as a white solid.

m.p. 151-152° C.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1H, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1H, s), 8.88 (1H, s), 15.44 (1H, s)

MS (ESI): M+ 448

Step 2

Compound A (32.3 g) obtained in Step 1 was dissolved in butyl acetate(160 ml) with heating under reflux. The crystal form II of Example 2 wasseeded at 63° C. and the mixture was stirred for 3 hr while allowing tocool. The precipitated solid was collected by filtration and vacuumdried to give the crystal of compound A (crystal form II) (24.79 g,yield 77%) as a white solid.

Example 2-5: Production of Crystal Form II of the Compound A

Step 1

1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (19 g, 33 mmol) obtained in Example 1, Step 5 wasdissolved in isopropanol (100 ml), 1N aqueous sodium hydroxide solution(200 ml, 200 mmol) was added, and the mixture was heated under refluxfor 2.5 hr. The reaction mixture was allowed to cool to roomtemperature, and the mixture was filtered through Celite. The filtratewas acidified by adding concentrated hydrochloric acid, and stirred atroom temperature for 2 hr. The precipitated solid was collected byfiltration and vacuum dried to give6-(3-chloro-2-fluorobenzyl)-7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (12 g, yield 82%) as a pale-yellow solid.

¹H NMR (DMSO-d₆ 400 MHz) (δ) ppm: 0.71 (3H, d, J=6.5 Hz), 1.13 (3H, d,J=6.5 Hz), 2.36 (1H, br), 3.77 (1H, br), 3.94 (1H, br), 4.25 (2H, s),4.77 (1H, br), 5.16 (1H, t, J=2.4 Hz), 7.19-7.23 (1H, m), 7.32-7.35 (1H,3), 7.48-7.52 (1H, m), 8.24-8.28 (2H, m), 9.00 (11H, s), 15.00 (1H, s)

Step 2

The compound (12 g, 27 mmol) obtained in Step 1 was dissolved inmethanol (64 ml), 28% sodium methoxide methanol solution (52 ml, 256mmol) was added, and the mixture was heated under reflux for 24 hr. Thereaction mixture was allowed to cool to room temperature and filteredthrough Celite. The filtrate was concentrated under reduced pressure.The residue was acidified by adding water (360 ml) and concentratedhydrochloric acid, and extracted with ethyl acetate. The organic layerwas washed successively with water and saturated brine and dried oversodium sulfate. The mixture was filtered, and the filtrate wasconcentrated under reduced pressure to give a crude product (13 g) as abrown oil. The obtained crude product (13 g) was dissolved in isobutylacetate (60 ml) by heating and, after seeding, the mixture was stirredfor 23 hr while allowing to cool. The precipitated solid was collectedby filtration, and vacuum dried to give compound A (9.2 g, yield 75%) asa white solid.

¹H MR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1H, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1H, s), 8.88 (1H, s), 15.44 (1H, s)

MS (ESI): M+ 448

Step 3

2-(2,4-Difluoro-5-iodobenzoyl)-3-dimethylaminoacrylic acid ethyl ester(20 g) obtained in Example 1, Step 2 was subject to slurry washing witha mixed solvent of ethyl acetate (60 ml) and hexane (40 ml) and heatedunder reflux. The mixture was filtered, and the remaining solid wasvacuum dried to give7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (18 g, yield 94%) as a beige solid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.6 Hz), 1.10 (3H, d,J=6.6 Hz), 1.28 (3H, t, J=7.0 Hz), 2.27 (1H, br), 3.77 (1H, br), 3.86(1H, br), 4.23 (2H, q, J=7.0 Hz), 4.56 (1H, br), 5.12 (1H, t, J=4.9 Hz),8.09 (1H, d, J=11.1 Hz), 8.62 (1H, d, J=7.5 Hz), 8.68 (1H, s)

MS(ESI): M+ 448

Step 4

The compound (19 g, 42 mmol) obtained in Step 3 was dissolved indimethylformamide (65 ml), imidazole (3.4 g, 49.9 mmol) andtert-butyldimethylsilyl chloride (7.2 g, 47.8 mmol) were added, and themixture was stirred at room temperature for 1.5 hr. Water was added tothe reaction mixture, and the mixture was extracted with ethyl acetate.The organic layer was washed successively with water, saturated aqueousammonium chloride solution and saturated brine, and dried over sodiumsulfate. The organic layer was filtered, and the filtrate wasconcentrated under reduced pressure to give a crude product (24 g) of1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester as a beige amorphous form.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.07 (3H, s), −0.05 (3H, s), 0.77 (9H,s), 0.84 (3H, d, J=6.5 Hz), 1.18 (3H, d, J=6.5 Hz), 1.40 (3H, t, J=7.2Hz), 2.35-2.50 (1H, m), 3.85-3.95 (1H, m), 3.98-4.10 (2H, m), 4.30-4.40(2H, m), 7.26 (1H, s), 8.64 (1H, s), 8.94 (1H, d, J=7.2 Hz)

MS (ESI): M+ 562

Step 5

The crude product (24 g) obtained in Step 4 was dissolved intetrahydrofuran (200 ml) and, under an argon stream,dibenzylidenacetonepalladium(II) (984 mg, 1.7 mmol) andtrifurylphosphine (795 mg, 3.4 mmol) were added, and a solution (56 ml,56 mmol) of 1M 3-chloro-2-fluorobenzylzinc bromide obtained in the samemanner as in Example 1, Step 5 in tetrahydrofuran was added dropwise at60° C. After the completion of the dropwise addition, the mixture wasstirred with heating at the same temperature for 2 hr. The reactionmixture was allowed to cool to room temperature, saturated aqueousammonium chloride solution was added, and filtered through Celite, andthe filtrate was extracted twice with ethyl acetate. The organic layerwas washed successively with water (twice) and saturated brine, anddried over magnesium sulfate. The organic layer was filtered, and thefiltrate was concentrated under reduced pressure to give a crude product(30 g) of1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester as a brown paste.

Step 6

The crude product (30 g) obtained in Step 5 was dissolved in isopropanol(150 ml), IN aqueous sodium hydroxide solution (300 ml, 300 mmol) wasadded, and the mixture was heated under reflux for 2.5 hr. The reactionmixture was allowed to cool to room temperature, and the mixture wasfiltered through Celite. The filtrate was acidified by addingconcentrated hydrochloric acid, and the mixture was stirred at roomtemperature for 2 hr. The precipitated solid was collected by filtrationand vacuum dried to give a crude product (18 g) as a beige solid. Theobtained crude product (18 g) was suspended in butyl acetate (90 ml),and subjected to slurry stirring with heating under reflux for 1 hr. Thesuspension was allowed to cool to room temperature, filtered and vacuumdried to give6-(3-chloro-2-fluorobenzyl)-7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (11 g, yield 62% (relative to Step 3)) as a white solid.

¹H NMR (DMSO-di 400 MHz) (δ) ppm: 0.71 (3H, d, J=6.5 Hz), 1.13 (3H, d,J=6.5 Hz), 2.36 (1H, br), 3.77 (1H, br), 3.94 (1H, br), 4.25 (2H, s),4.77 (1H, br), 5.16 (1H, t, J=2.4 Hz), 7.19-7.23 (1H, m), 7.32-7.35 (1H,m), 7.48-7.52 (1H, m), 8.24-8.28 (2H, m), 9.00 (1, s), 15.00 (1H, s)

MS(ESI): M+ 436

Step 7

The compound (11 g, 26 mmol) obtained in Step 6 was dissolved inmethanol (60 ml), 28% sodium methoxide methanol solution (52 ml, 256mmol) was added, and the mixture was heated under reflux for 24 hr. Thereaction mixture was allowed to cool to room temperature and filteredthrough Celite, and the filtrate was concentrated under reducedpressure. The residue was acidified by adding water (330 ml) andconcentrated hydrochloric acid, and the mixture was extracted with ethylacetate. The organic layer was washed successively with water andsaturated brine, and dried over sodium sulfate. The mixture wasfiltered, and the filtrate was concentrated under reduced pressure togive a crude product (12 g) as a brown oil. The obtained crude product(12 g) was dissolved in isobutyl acetate (60 ml) by heating underreflux. A seed crystal (crystal form II of compound A) was seeded, andthe mixture was stirred for 23 hr while allowing to cool. Theprecipitated solid was collected by filtration and vacuum dried to givecompound A (8.2 g, yield so 71%) as a white solid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.5 Hz), 1.16 (3H, d,J=6.5 Hz), 2.30-2.50 (1H, m), 3.70-3.90 (1H, m), 3.90-4.00 (1H, m), 4.03(3H, s), 4.12 (2H, s), 4.80-4.90 (1H, m), 5.19 (1H, t), 7.19-7.25 (2H,m), 7.46-7.51 (2H, m), 8.04 (1H, s), 8.88 (1H, s), 15.44 (1H, s)

MS (ESI): M+ 448

Step 8

Compound A (7.66 g) obtained in Step 7 and compound A (9.17 g) obtainedin Step 2 were dissolved in isobutyl acetate (84 ml) by heating underreflux and the mixture was stirred for 16 hr while allowing to cool. Theprecipitated solid was collected by filtration and vacuum dried to givecrystal form II (14.73 g, yield 88%) of compound A as a white solid.

Example 2-6: Production of Crystal Form II of the Compound A

Step 1

2,4-Difluorobenzoic acid (100 g, 633 mmol) was dissolved intrifluoromethanesulfonic acid (400 ml) and N-iodosuccinimide (157 g, 696mmol) was added by portions at not more than 5° C. After the completionof the addition, the mixture was stirred at 50° C. for 1.5 hr. Thereaction mixture was poured into iced water, and the mixture was stirredfor 1 hr. The precipitated solid was collected by filtration, washedsuccessively with water and hexane, and vacuum dried to give2,4-difluoro-5-iodobenzoic acid (179 g yield quantitative) as a whitesolid.

¹H NMR (CDCl₃ 300 MHz) (σ) ppm: 6.94 (1H, dd, J=10.3, 10.3 Hz), 8.46(1H, d, J=7.5 Hz)

Step 2

The compound (28 g, 100 mmol) obtained in Step 1 was dissolved in ethylacetate (300 ml), oxalyl chloride (11 ml, 122 mmol) anddimethylformamide (catalytic amount) were added, and the mixture wasstirred at room temperature for 2 hr. The filtrate was concentratedunder reduced pressure and azeotroped with toluene. The residue wasdissolved in tetrahydrofuran (100 ml), this solution was added dropwiseto a solution of ethyl 3,3-dimethylaminoacrylate (17 g, 120 mmol) andtriethylamine (21 ml, 150 mmol) in tetrahydrofuran (100 ml), and themixture was heated under reflux for 3 hr. The reaction mixture wasallowed to cool and ethyl acetate (200 ml) was added. The mixture waswashed successively with water (twice) and saturated brine, and driedover sodium sulfate. The mixture was filtered and the filtrate wasconcentrated under reduced pressure. The obtained residue was subject toslurry stirring with a mixed solvent of diethyl ether (50 ml) and hexane(50 ml). The mixture was filtered and the remaining solid was vacuumdried to give a crude product (26 g, yield 63%) of2-(2,4-difluoro-5-iodobenzoyl)-3-dimethylaminoacrylic acid ethyl esteras a yellow solid.

Step 3

The crude product (22 g, 55 mmol) obtained in Step 2 was dissolved intetrahydrofuran (110 ml), (S)-(+)-valinol (6.8 g, 65.8 mmol) was added,and the mixture was stirred with heating at 50° C. for 30 min. Thereaction mixture was concentrated under reduced pressure, and theresidue was dissolved in ethyl acetate (100 ml), washed successivelywith water and saturated brine, and dried over magnesium sulfate. Themixture was filtered and the filtrate was concentrated under reducedpressure. The obtained residue was dissolved in dimethylformamide (80ml), potassium carbonate (19 g, 137 mmol) was added, and the mixture wasstirred with heating at 60° C. for 1.5 hr. The reaction mixture wasallowed to cool to room temperature and concentrated under reducedpressure. Water (250 ml) was added to the obtained residue and themixture was stirred at room temperature for 30 min. The precipitatedsolid was collected by filtration.

The obtained solid was washed successively with a mixed solvent of water(100 ml), ethyl acetate (10 ml) and hexane (40 ml) and vacuum dried togive7-fluoro-1-((S)-1-hydroxymethyl-2-methylpropyl)-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (22 g, yield 88%) as a pale-yellow solid.

¹H NMR (DMSO-d₆ 300 MHz) (δ) ppm: 0.72 (3H, d, J=6.6 Hz), 1.10 (3H, d,J=6.6 Hz), 1.28 (3H, t, J=7.0 Hz), 2.27 (1H, br), 3.77 (1H, br), 3.86(1H, br), 4.23 (2H, q, J=7.0 Hz), 4.56 (1H, br), 5.12 (1H, t, J=4.9 Hz),8.09 (1H, d, J=11.1 Hz), 8.62 (1H, d, J=7.5 Hz), 8.68 (1H, s)

MS(ESI): M+ 448

Step 4

The compound (22 g, 48 mmol) obtained in Step 3 was dissolved indimethylformamide (60 ml), imidazole (3.9 g, 57.7 mmol) andtert-butyldimethylsilyl chloride (8.0 g, 53.0 mmol) were added, and themixture was stirred at room temperature for 1.5 hr. The reaction mixturewas concentrated under reduced pressure, and the obtained residue wasdissolved in ethyl acetate (200 ml), washed successively with water(twice) and saturated brine, and dried over sodium sulfate. The mixturewas filtered and the filtrate was concentrated under reduced pressure.The obtained residue was purified by silica gel chromatography (ethylacetate:hexane-3:7 to 4:6) to give1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-7-fluoro-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (25 g, yield 92%) as a white wax.

¹H NMR (CDCl₃ 4000 MHz) (δ) ppm: −0.070 (3H, s), −0.05 (3H, s), 0.77(9H, s), 0.84 (3H, d, J=6.5 Hz), 1.18 (3H, d, J=6.5 Hz), 1.40 (3H, t,J=7.2 Hz), 2.35-2.50 (1H, m), 3.85-3.95 (1H, m), 3.98-4.10 (2H, M),4.30-4.40 (2H, m), 7.26 (1H, s), 8.64 (1H, s), 8.94 (1H, d, J=7.2 Hz)

MS (ESI): M+ 562

Step 5

The compound (25 g, 44 mmol) obtained in Step 4 was dissolved intetrahydrofuran (200 ml) and, under an argon stream,dibenzylidenacetonepalladium(II) (1.0 g, 1.8 mmol) and trifurylphosphine(824 mg, 3.5 mmol) were added. A solution (58 ml, 58 mmol) of 1M3-chloro-2-fluorobenzylzinc bromide obtained in the same manner as inExample 1, Step 5 in tetrahydrofuran was added dropwise at 60° C. Afterthe completion of the dropwise addition, the mixture was heated underreflux for 3 hr. The reaction mixture was allowed to cool to roomtemperature, ethyl acetate (200 ml) was added, washed successively with1N hydrochloric acid, water, saturated aqueous sodium hydrogencarbonate, water and saturated brine, and dried over sodium sulfate. Themixture was filtered and the filtrate was concentrated under reducedpressure. The obtained residue was purified by silica gel chromatography(ethyl acetate:hexane-4:6 to 1:1) to give1-((S)-1-tert-butyldimethylsilyloxymethyl-2-methylpropyl)-6-(3-chloro-2-fluorobenzyl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid ethyl ester (17 g, yield 68%) as a pale-yellow oil.

¹H NMR (CDCl₃ 400 MHz) (δ) ppm: −0.09 (3H, s), −0.05 (3H, s), 0.75 (9H,U), 0.85 (3H, d, J=6.7 Hz), 1.18 (3H, d, 6.7 Hz), 1.39 (3H, t, J=7.1Hz), 2.45 (1H, br), 3.89-3.92 (1H, m), 3.98-4.02 (1l, m), 4.07-4.12 (1H,m), 4.12 (2H, s), 4.34-4.41 (2H, m), 6.96-7.00 (1H, m), 7.03-7.05 (1H,m), 7.21-7.24 (1H, m), 7.26-7.29 (1H, m), 8.39 (1H, d, J=8.8 Hz), 9.63(1H, s)

Step 6

The compound (17 g, 30 mmol) obtained in Step 5 was dissolved inmethanol (120 ml), 28% sodium methoxide methanol solution (62 ml, 304mmol) was added, and the mixture was heated under reflux for 19 hr. Thereaction mixture was allowed to cool to room temperature, water (200 ml)was added, and methanol was evaporated under reduced pressure. Theresidue was acidified by adding concentrated hydrochloric acid,extracted with ethyl acetate, and dried over sodium sulfate. The mixturewas filtered, and the filtrate was concentrated under reduced pressureto give a crude product (14 g) of compound A as a pale-yellow oil.

Step 7

Compound A (14.11 g) obtained in Step 6 was suspended in a mixed solventof ethyl acetate (20 ml) and hexane (20 ml) at room temperature, a seedcrystal (crystal form II of compound A) was seeded, and the mixture wasstirred for 1 hr. The suspension was filtered, and the remaining solidwas vacuum dried to give compound A (crystal form II, 10.40 g, yield77%) as a white solid.

Example 3: Production of Crystal Form III of the Compound A Example 3-1:Production of Crystal Form III of the Compound A

The crystal form II of compound A (10.0 g, 22.3 mmol) obtained inExample 2-2 was added to isobutyl acetate (30 mL) and the crystal wasdissolved by heating under reflux. The solution was cooled to 90° C.,and stirred for 5 hr to allow precipitation of crystals. This solutionwas further allowed to cool to room temperature, and further stirred for12 hr. The precipitated crystal was collected by filtration. Theobtained crystal was washed with isobutyl acetate (10 mL) and vacuumdried to give a white crystal (9.85 g, yield 98.5%). Since this crystalwas confirmed to be different from crystal form II by XRPD analysis, acrystal showing the XRPD chart (FIG. 1) as this crystal was taken ascrystal form III.

Example 3-2: Production of Crystal Form III of the Compound A

The crystal form II of compound A (250 g, 558 mmol) obtained in Example2-2 was added to isobutyl acetate (750 mL). A seed crystal (12.5 g) ofthe crystal form III of compound A obtained in Example 3 was added atroom temperature and the mixture was stirred for 17 hr. The precipitatedcrystal was collected by filtration. The obtained crystal was washedwith isobutyl acetate (250 mL), and vacuum dried to give the objectproduct (crystal form III, 259 g, yield 98.6%) as a white crystal. Thiscrystal was confirmed to be equivalent to the standard product of thecrystal (crystal form III of compound A obtained in Example 3-1) by XRPDanalysis.

Example 3-3: Production of Crystal Form III of the Compound A

Compound A (crystal form II, 10.0 g, 22.3 mmol) obtained in Example 2-2was added to isopropanol (30 mL), and the mixture was heated underreflux to dissolve the crystal. The solution was cooled to 70° C., aseed crystal (10 mg) of the crystal form III of compound A obtained inExample 3-1 was added, and the mixture was stirred for 5 hr. Thismixture was further allowed to cool to room temperature, stirred for 12hr, and the crystal was collected by filtration. The obtained crystalwas washed with isopropanol (10 mL), and vacuum dried to give the objectproduct (crystal form III, 9.72 g, yield 97.2%) as a white crystal. Thiscrystal was confirmed to be equivalent to the standard product of thecrystal (crystal form III of compound A obtained in Example 3-1) by XRPDanalysis.

Example 3-41 Production of crystal form III of compound A The crystalform II of compound A (7.00 g, 15.6 mmol) obtained in Example 2-2 wasadded to a mixed solution of ethanol (52.5 mL) and water (7 mL) anddissolved by heating. Water (28 mL) was added, a seed crystal (10 mg) ofthe object product was added at 70° C. and the mixture was stirred for 4hrs. After allowing to cool to room temperature, the mixture wasice-cooled and further stirred for 2 hrs, and the crystals werecollected by filtration. The obtained crystals were washed with a mixedsolution of cool ethanol (8.4 mL) and water (5.6 mL) and vacuum dried togive the object product as white crystals (crystal form III, 6.77 g,yield 96.8%). This crystal was confirmed to be equivalent to thestandard product (Example 3-1) by XRPD analysis.

Experimental Example

The property values of each crystal form were determined by thefollowing analysis tests, and the stability test of each crystal formwas performed using them as indices.

Sample

Unless otherwise specified, the aforementioned crystal (crystal form I)obtained in Reference Example 1, the crystal (crystal form II) obtainedin Example 1 and the crystal (crystal form III) obtained in Example 3-1were used as samples.

Analysis Test

1. X-Ray Powder Diffractometry

This test aims at obtaining X-ray powder diffraction patterns to specifythe crystal form of the crystals obtained in Reference Example 1,Example 1 and Example 3-1. The diffraction patterns are utilized tospecify the crystal form, evaluate the stability, to determine thepurity and the like.

A sample was fixed to an aluminum cell, and the measurement wasperformed using an X-ray powder diffractometer (RINT 2000/PC Ultima⁺,manufactured by Rigaku Corporation, X-ray source: Cu-Kα1 ray, tubevoltage: 40 kV, tube electric current: 40 mA, scan speed: 5° per min,step width: 0.02°, diffraction angle: 5-40°), based on which thediffraction patterns were obtained. The obtained diffraction patternsare shown in FIG. 1.

As shown in FIG. 1, X-ray powder diffraction patterns obtained fromrespective samples were different.

Therefore it was confirmed that the crystals obtained in ReferenceExample 1, Example 1 and Example 3-1 were distinct from each other, andshow characteristic diffraction patterns as shown in the X-ray powderdiffraction pattern. Therefore, in the present specification, they werenamed as crystal form I, crystal form II and crystal form III, based onthese X-ray 23 powder diffraction patterns.

For specification of the crystal form, the diffraction peakcharacteristic of each crystal may be evaluated in a comprehensivemanner based on the diffraction chart in FIG. 1.

The main diffraction peaks and characteristic diffraction peaksspecified from the diffraction patterns in FIG. 1 are shown below.

Crystal Form I

Main diffraction peak:2θ=6.58, 14.40, 14.64, 15.24, 16.48, 19.16, 20.90,21.14, 22.24, 24.74, 25.64, 26.12, 27.20°;

Characteristic diffraction peak:2θ=6.58, 14.40, 19.16, 20.90, 21.14°.

Crystal Form II

Main diffraction peak:2θ=6.56, 9.04, 13.20, 14.62, 15.24, 16.48, 19.86,20.84, 21.22, 22.24, 25.22, 25.96, 26.12, 27.34°;

Characteristic diffraction peak:2θ=6.56, 13.20, 19.86, 20.84, 21.22,25.22°.

Crystal Form III

Main diffraction peak:2θ=8.54, 14.02, 15.68, 15.90, 16.00, 17.06, 17.24,17.84, 18.12, 19.50, 19.90, 22.26, 22.68, 23.02, 24.16, 24.76, 25.18,25.74, 25.98, 27.50, 28.80, 30.38, 30.72, 32.54°;

Characteristic diffraction peak:2θ=8.54, 14.02, 15.68, 17.06, 17.24,24.16, 25.74°.

2. Thermal Analysis

This test aims at the measurement of the enthalpy and extrapolated onsettemperature at an endothermic peak on the Differential ScanningCalorimetry (DSC) measurement curve. These values are among the indicesof the stability of the above-mentioned crystal form I, crystal form IIand crystal form III, and can be used as an index to specify the crystalform.

2.1. Enthalpy and Extrapolated Onset Temperature of Crystal Form I andCrystal Form II

Crystal form I and crystal form II were subjected to measurement using aDifferential Scanning Calorimetry (DSC) measurement apparatus (DSC8240,manufactured by Rigaku Corporation), under atmosphere, measurementsample 5±1 mg, temperature rise rate: 10° C./min, aluminum open pan, andalumina oxide as a reference. The enthalpy and extrapolated onsettemperature at an endothermic peak on the obtained DSC curve weredetermined.

2.2. Enthalpy and Extrapolated Onset Temperature of Crystal Form III

Crystal form III was subjected to measurement using a DSC measurementapparatus (DSC8240, manufactured by Rigaku Corporation), underatmosphere, measurement sample 5.0±0.5 mg, temperature rise rate: 5°C./min, aluminum closed pan, and alumina oxide as a reference. Theenthalpy and extrapolated onset temperature at an endothermic peak onthe obtained DSC curve were determined.

The results are shown in Table 1.

TABLE 1 DSC curve at endothermic peak on enthalpy and extrapolated onsettemperature Endothermic peak Crystal Extrapolated onset form Enthalpy(J/g) temperature (° C.) Crystal form I 51.080 150.3 Crystal form II53.542 151.2 Crystal form III 81.404 162.1

As shown in Table 1, crystal form III shows greatest enthalpy andhighest extrapolated onset temperature among three crystal forms crystalform I. Thus, crystal form III was confirmed to be most stable form.

3. Purity Test

This test aims at measurement of the purity of compound A. The puritycan be used as indices of chemical stability.

3.1. Purity of Compound of Crystal Form I and Crystal Form II

Each sample (crystal form I and crystal form II, ca. 10 mg) wasdissolved in acetonitrile to make an amount of 10 mL and used as asample solution. This solution (10 μL) was applied to high performanceliquid chromatography (HPLC) under the following conditions. The peakarea of each sample solution was measured by automatic integration, andthe purity was determined by the following formula. The purity is shownin the Tables 5 and 6 below.

Purity (%)=100−(A _(sum) /A _(s))×100

As: total peak area of peaks obtained from sample solution

Asum: total peak area of peaks other than the main peak obtained fromsample solution

Test Conditions

Detector: UV absorptiometer (wavelength: 259 nm)

Column: CAPCELL PAX MG (inner diameter 4.6 cm, length 15 cm, particlesize 5 μm, manufactured by Shiseido Co., Ltd.

Column temperature: constant temperature around 40° C.

Mobile phase A: trifluoroacetic acid solution (1:1000)

Mobile phase B: solution of trifluoroacetic acid in acetonitrile(1:1000)

Gradient program: As shown in the following Table 2, the mixing ratio ofmobile phase A and mobile phase B is changed to control concentrationgradient.

TABLE 2 Time (min) Mobile Mobile after injection phase A (%) phase B (%)0 55 45 0-5 55→52 45→48  5-15 52 48 15-25 52→20 48→80 25-35 20 80 35-3620→55 80→45 36-45 55 45

Flow rate: 1 mL/min

3.2. Purity of Compound of Crystal Form III

A sample (crystal form III, ca. 50 mg) was dissolved in a mixture (4:1)of mobile phase B and mobile phase A to make an amount of 50 mL, whichwas used as a sample solution. This solution (1 mL) was preciselymeasured and a mixture (4:1) of mobile phase B and mobile phase A wasadded to precisely make an amount of 100 mL, which was used as astandard solution. The sample solution and standard solution (15 μL) wasapplied to high performance liquid chromatography (HPLC) under thefollowing conditions. The peak area of each solution was measured byautomatic integration, and the purity was determined by the followingformula. The purity is shown in the Table 7 below.

Purity (%)=100−(A _(sum) /A _(r))

A_(r): peak area of main peak obtained from standard solution

A_(sum): total peak area of peaks other than the main peak go obtainedfrom sample solution

Test Conditions

Detector: UV absorptiometer (wavelength: 259 nm)

Column: Waters XTerra MC C18 (inner diameter 4.6 cm, length 5 cm,particles diameter 2.5 μm, manufactured by Waters)

Column temperature: constant temperature around 40° C.

Mobile phase A: phosphoric acid is added to dipotassiumhydrogenphosphate solution (1-41149) to adjust pH to 7.0

Mobile phase B: acetonitrile

Gradient program: As shown in the following Table 3, the mixing ratio ofmobile phase A and mobile phase B is changed to control concentrationgradient.

TABLE 3 Time (min) Mobile Mobile after injection phase A (%) phase B (%) 0-15 58 42 15-35 58→20 42→80 35-45 20 80 45-46 20→58 80→42 46-55 58 42

Flow rate: 0.9 mL/min

4. Solubility Test

This test aims at measurement of solubility of the crystal in varioustest solutions and under various pHs. The solubility is one of theindices of the stability of the above-mentioned crystal form I, crystalform II and crystal form III and can be used also as a reference indicesof absorbability of crystal form by living organisms.

Each sample (crystal form I type I, crystal form II and crystal formIII, ca. 10 mg) was placed in a 10 L centrifuge tube together with thefollowing test solution (5 mL) and shaken with a shaker (SR-1M;manufactured by Tietech Co., Ltd.) for 14 hours. After shaking, themixture was centrifuged (3000 rpm, 20 min) and the supernatant warnfiltered through 0.2 m pore size −13 mm diameter polytetrafluoroethylenedisk filter (Millex-LG; manufactured by Millipore Corporation). Themeasurement war performed by high performance liquid chromatography(HPLC). The results are shown in Table 4.

TABLE 4 Solubility Test Solubility (μg/mL) Crystal form Crystal formTest solution pH II III Purified — 0.5 <0.1 water Japanese 1.2 0.9 <0.1Pharmacopoeia 1st fluid ¹⁾ Japanese 6.8 5.8 2.4 Pharmacopoeia 2nd fluid²⁾ McIlvaine ³⁾ 2.2 1.9 <0.1 4 0.8 <0.1 5 0.4 <0.1 6 1.0 0.7 6.8 6.3 0.78 84 23 ¹⁾ Japanese Pharmacopoeia, General Test Method, DisintegrationTest Method, 1st fluid. Hydrochloric acid (7.0 mL) and water are addedto sodium chloride (2.0 g) to make an amount of 1000 mL. This solutionis transparent and colorless and has a pH of about 1.2. ²⁾ JapanesePharmacopoeia, General Test Method, Disintegration Test Method, 2ndfluid. 0.2 mol/L sodium hydroxide sample (118 mL) and water are added to0.2 mol/L potassium disodium phosphate sample (250 mL) to make an amountof 1000 mL. This solution is transparent and colorless and has a pH ofabout 6.8. ³⁾ McIlvaine buffer obtained by mixing disodiumhydrogenphosphate and citric acid at a given ratio to adjust to a givenpH.

From the above-mentioned results, it was confirmed that crystal form IIhas higher solubility than crystal form III.

5. Stability Test

A stability test of each sample was performed under the followingpreservation conditions. The results of crystal form I are shown inTable 5, the results of crystal form II are shown in Table 6, and theresults of crystal form III are shown in Table 7.

As shown in Table 6 and Table 7, crystal form II and crystal form IIIdid not show any difference in the test results under all preservationconditions, as compared to the initial sample. In contrast, as shown inTable 5, crystal form I showed changes in the X-ray powder diffractionpattern obtained from sample after preservation under preservationcondition #3 (80° C., preservation in an open container for 3 days) andpreservation condition #5 (60° C., preservation in an open container for3 weeks), and the X-ray powder diffraction pattern of crystal form I wasobserved to have overlapped with the X-ray powder diffraction patternderived from crystal form II. Thus, it was evaluated that a part of thesample showed crystal transition to crystal form II during preservation.The X-ray powder diffraction patterns of the samples preservation sampleunder preservation conditions #1-6 of crystal form I are shown in FIG.2.

TABLE 5 Results of stability test of crystal form I Preservation Thermalconditions Appearance Purity XRD analysis 1 Initial — 99.1% DiffractionEndothermic pattern of peak crystal (extrapolated form I onsettemperature 150.3° C.) 2 80° C. No change 99.1% No change No change inClosed in in DSC curve container appearance diffraction Preservedpattern for 3 days 3 80° C. No change 99.1% Overlap of No change in Openin diffraction DSC curve container appearance patterns of Preservedcrystal for 3 form I and days crystal form II 4 60° C. No change 99.1%No change No change in Closed in in DSC curve container appearancediffraction Preserved pattern for 3 weeks 5 60° C. No change 99.1%Overlap of No change in Open in diffraction DSC curve containerappearance patterns of Preserved crystal for 3 form I and weeks crystalform II 6 60° C./75% No change 99.1% No change No change in R.H. in inDSC curve Open appearance diffraction container pattern Preserved for 3weeks R.H.: relative humidity XRD: X-ray powder diffractometry

TABLE 6 Results of stability test of crystal form II PreservationThermal conditions Appearance Purity XRD analysis 1 Initial — 98.9%Diffraction Endothermic pattern of peak crystal (extrapolated form IIonset temperature 151.2° C.) 2 80° C. No change 98.9% No change Nochange in Closed in in DSC curve container appearance diffractionPreserved pattern for 3 days 3 80° C. No change 98.8% No change Nochange in Open in in DSC curve container appearance diffractionPreserved pattern for 3 days 4 60° C. No change 98.9% No change Nochange in Closed in in DSC curve container appearance diffractionPreserved pattern for 3 weeks 5 60° C. No change 98.8% No change Nochange in Open in in DSC curve container appearance diffractionPreserved pattern for 3 weeks 6 60° C./75% No change 98.9% No change Nochange in R.H. in in DSC curve Open appearance diffraction containerpattern Preserved for 3 weeks R.H.: relative humidity XRD: X-ray powderdiffractometry

TABLE 7 Results of stability test of crystal form III PreservationThermal conditions Appearance Purity XRD analysis 1 Initial — 98.71%Diffraction Endothermic pattern of peak crystal (extrapolated form IIIonset temperature 162.1° C.) 2 80° C. No change 98.68% No change Nochange in Closed in in DSC curve container appearance diffractionPreserved pattern for 3 days 3 80° C. No change 98.69% No change Nochange in Open in in DSC curve container appearance diffractionPreserved pattern for 3 days 4 60° C. No change 98.67% No change Nochange in Closed in in DSC curve container appearance diffractionPreserved pattern for 3 weeks 5 60° C. No change 98.66% No change Nochange in Open in in DSC curve container appearance diffractionPreserved pattern for 3 weeks 6 60° C./75% No change 98.65% No change Nochange in R.H. in in DSC curve Closed appearance diffraction containerpattern Preserved for 3 weeks R.H.: relative humidity XRD: X-ray powderdiffractometry

From the results of the above-mentioned stability test, it was observedthat the crystal form I was unstable but crystal form II and crystalform III were extremely stable under various preservation conditions.Therefore, it was evidenced that crystal form II and crystal form IIIare preferable for use as a pharmaceutical product and the like.

As for the absorbability by living organisms, crystal form II is morepreferable, and crystal form III is more preferable because it is themost stable crystal.

Since crystal form II and crystal form III are both stable, a mixedcrystal of them can be used for the present invention.

Experimental Example

The following explains evaluation methods of the HIV integraseinhibitory activity of a crystal or a mixed crystal of compound A of thepresent invention.

(i) Construction of Recombinant Integrase Gene Expression System

The 185th phenylalanine of HIV integrase full length gene (J. Virol.,67, 425-437 (1993)) was substituted by histidine and inserted into therestriction enzyme NdeI and XhoI sites of the plasmid pET21a (+)(manufactured by Novagen), whereby an integrase expression vectorpET21a-IN-F185H was constructed.

(ii) Production and Purification of Integrase Protein

Escherichia coli recombinant BL21 (DE3) transformed with plasmidpET21a-IN-F185H obtained in (i) was shake cultured at 30° C. in a liquidmedium containing ampicillin. When the culture reached the logarithmicgrowth phase, isopropyl-β-D-thiogalactopyranoside was added to promoteexpression of integrase gene. The culture was continued for 3 hr topromote accumulation of the integrase protein. The recombinant E. coliwas collected in pellets by centrifugal separation and preserved at −80°C.

The E. coli was suspended in Lysis buffer (20 mM HEPES (pH 7.5), 5 mMDTT, 10 mM CHAPS, 10% glycerol) containing IM sodium chloride andsubjected to repeat pressurization and depressurization for rupture, andcentrifugal separation at 4° C., 40,000×g, 60 min to recover awater-soluble fraction (supernatant). This was diluted 10-fold withLysis buffer free of sodium chloride, mixed with SP-Sepharose(manufactured by Pharmacia Corporation) and stirred at 4° C. for 60 minto allow adsorption of integrase protein to the resin. The resin waswashed with Lysis buffer containing 100 mM sodium chloride and theintegrase protein was eluted with Lysis buffer containing 1M sodiumchloride.

The eluted integrase protein solution was applied to a Superdex 75(Pharmacia Corporation) column for gel filtration. The protein waseluted with Lysis buffer containing 1M sodium chloride.

The obtained fractions of the integrase protein were collected andpreserved at −80° C.

(iii) Preparation of DNA Solution

The following DNA synthesized by Greiner was dissolved in TE buffer (10mM Trim-hydrochloric acid (pH 8.0), 1 mM EDTA) and mixed with donor DNA,target DNA, and each complementary strand (+ and − strands) to 1 μM. Themixture was heated at 95° C. for 5 min, 80° C. for 10 min, 70° C. for 10min, 60° C. for 10 min, 50° C. for 10 min and 40° C. for 10 min andpreserved at 25° C. to give a double stranded DNA, which was used forthe test.

Donor DNA (− strand having biotin attached to the 5′ terminal)Donor + strand: (SEQ ID NO: 1) 5′-Biotin-ACC CTT TTA GTC AGT GTG GAA AATCTC TAG CA-3′ Donor - strand: (SEQ ID NO: 2)5′-ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA G-3′Target DNA (+, − strands both having digoxigenin added at 3′ terminal)Target + strand (SEQ ID NO: 3) 5′-TGA CCA AGG GCT AAT TCA CT-Dig-3′Target - strand: (SEQ ID NO: 4) 5′-AGT GAA TTA GCC CTT GGT CA-Dig-3′

(iv) Determination of Enzyme (HIV Integrase) Inhibitory Activity

The donor DNA was diluted with TE buffer to 10 nM, of which 50 μl wasadded to each well of streptavidin-coated microtiter plate (manufacturedby Roche) and allowed to adsorb at 37° C. for 60 min. The DNA was thenwashed with phosphate buffer (Dulbecco PBS, Sanko Junyaku Co., Ltd.)containing 0.1% Tween 20 and phosphate buffer. Then, a reaction mixture(70 μl, see the following * for the composition), a test substance (10μl) diluted with the reaction mixture and 100 μg/ml integrase protein(10 μl) were added to each well and reacted at 37° C. for 60 min. Then,50 nM target DNA (10 μl) was added, reacted at 37° C. for 10 min andwashed with phosphate buffer containing 0.1% Tween 20 to stop thereaction.

Then, 100 mU/ml peroxidase labeled anti-digoxigenin antibody solution(manufactured by Roche, 100 μl) was added, and the mixture was reactedat 37° C. for 60 min, followed by washing with phosphate buffercontaining 0.1% Tween 20.

A peroxidase color solution (manufactured by Bic Rad, 100 μl) was addedand allowed to react at room temperature for 4 min. The color reactionwas stopped by adding 1N sulfuric acid (100 μl). The absorbance at 450nm was measured.

The HIV integrase inhibitory activity (IC₅₀) of the compound A of thepresent invention was calculated from the inhibition rate according tothe following formula. The results are shown in Table 8.

Inhibition rate (%)=[1−(Object−Blank)/(Control−Blank)]×100

Object; absorbance of well in the presence of test compound

Control; absorbance of well in the absence of test compound

Blank; absorbance of well in the absence of test compound, in theabsence of integrase protein

*Composition of the reaction mixture: 30 mM morpholinopropanesulfonicacid (MOPS), 5 mM MgCl₂, 3 mM dithiothreithol (DTT), 0.1 mg/mL bovineserum albumin (BSA), 5S glycerol, 10% dimethyl sulfoxide (DMSO), 0.01%Tween 20

TABLE 8 Compound No. Enzyme activity IC₅₀ (μM) Compound A 0.0029

Evaluation of Antivirus Activity

The effect of combined use of a crystal or a mixed crystal of compound Aof the present invention and existent anti-HIV agents can be determinedin the following manner.

For example, the effect of combined use of two agents from existentnucleoside reverse transcriptase inhibitors (zidovudine, lamivudine,tenofovir), non-nucleoside reverse transcriptase inhibitors (efavirenz)or protease inhibitors (indinavir, nelfinavir) and a crystal or a mixedcrystal of compound A and the like are evaluated by XTT method usingCEM-SS cells infected with HIV-1 IIIB.

In addition, the effect of combined use of three agents of a crystal ora mixed crystal of compound A, zidovudine and lamivudine, or a crystalor a mixed crystal of compound A, tenofovir and lamivudine, and the likeis evaluated.

Prior to the combined use test, IC₅₀ and CC₅₀ of each pharmaceuticalagent alone are measured. 5 concentrations of pharmaceutical agent a and9 concentrations of pharmaceutical agent b, determined based on theseresults, are combined to evaluate the effect of combined use of twoagents. For combined use of three agents, a high concentrationpharmaceutical agent b and a pharmaceutical agent c are mixed andpharmaceutical agent a and the concentration are combined forevaluation.

The test results of the crystal or mixed crystal of compound A andcombination drug alone or in combination thereof are analyzed based onthe programs of Prichard and Shipman MacSynergy II version 2.01 andDeltagraph version 1.5d.

A three-dimensional plot is drawn from t inhibition at theconcentrations of each combined pharmaceutical agent, obtained from 3times of tests, with 95% (or 68%, 99%) confidence limits, and the effectof the combined use is evaluated based on the numerical values of μM²%calculated therefrom. The criteria of evaluation are shown in thefollowing.

Definition of interaction μM²% Strong synergistic action >100 Slightsynergistic action  +51-+100 Additive action +50-−50 Slight antagonisticaction  −51-−100 Strong antagonistic action <−100

Industrial Field of Utilization

The crystal of compound A of the present invention, which has theabove-mentioned particular crystal form, shows an anti-HIV effect aswell as superior crystal stability. Therefore, it is useful as astarting material of a pharmaceutical composition, particularly, variouspharmaceutical compositions for the prophylaxis and/or treatment ofAIDS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows multiple records of X-ray powder diffraction patterns,wherein the upper line shows the diffraction pattern of crystal formIII, the middle line shows the diffraction pattern of crystal form I,the lower line shows the diffraction pattern of crystal form II, thevertical axis shows diffraction intensity (cps: counts per second:intervals of scale is 2500 cps) and the transverse axis showsdiffraction angle 2θ(°).

FIG. 2 shows multiple records of X-ray powder diffraction patternobtained from the sample after 3-day preservation of the stability testof crystal form I. For comparison, the uppermost line shows thediffraction pattern (initial conditions) of crystal form II, and thelowermost line shows the diffraction pattern (initial conditions) ofcrystal form I. Shown from the second line are diffraction patternsunder preservation condition #6 (60° C./75% R.H., open container, 3weeks preservation), preservation condition #4 (60° C., container withstopper, 3 weeks preservation), preservation condition #5 (60° C., opencontainer, 3 weeks preservation), preservation condition #3 (80° C.,open container, 3 days preservation) and preservation condition #2 (80°C., container with stopper, 3 days preservation). The vertical axisshows diffraction intensity (cps: counts per second: intervals of scaleis 2500 cps) and the transverse axis shows diffraction angle 2θ(°).

Sequence Listing Free Text

SEQ ID; No 1: Donor+chain for determining HIV integrase activity

SEQ ID; No 2: Donor−chain for determining HIV integrase activity

SEQ ID; No 3: Target+chain for determining HIV integrase activity

SEQ ID; No 4: Target−chain for determining HIV integrase activity

The instant application includes a Statement Accompanying SequenceListing, and a Sequence Listing in both paper and computer-readableformats.

1-14. (canceled)
 15. A crystal form of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-meth-oxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid, having an X-ray powder diffraction pattern comprising acharacteristic diffraction peak at 6.56±0.2° 2θ.
 16. The crystal form ofclaim 15, having an X-ray powder diffraction pattern comprising acharacteristic diffraction peak at 6.56±0.1° 2θ.
 17. The crystal form ofclaim 15, having an X-ray powder diffraction pattern comprising acharacteristic diffraction peak at 6.56±0.06° 2θ.
 18. The crystal formof claim 15, having an X-ray powder diffraction pattern furthercomprising a characteristic diffraction peak at 21.22±0.2° 2θ.
 19. Thecrystal form of claim 16, having an X-ray powder diffraction patterncomprising characteristic diffraction peaks at 6.56±0.1° and 21.22±0.1°2θ.
 20. The crystal form of claim 17, having an X-ray powder diffractionpattern comprising characteristic diffraction peaks at 6.56±0.06°. and21.22±0.06° 2θ.
 21. The crystal form of claim 18, having an X-ray powderdiffraction pattern further comprising a characteristic diffraction peakat 13.20±0.2° 2θ.
 22. The crystal form of claim 19, having an X-raypowder diffraction pattern comprising characteristic diffraction peaksat 6.56±0.1°, 13.20±0.1°, and 21.22±0.1° 2θ.
 23. The crystal form ofclaim 21, having an X-ray powder diffraction pattern comprisingcharacteristic diffraction peaks at 6.56±0.06°, 13.20±0.06°, and21.22±0.06° 2θ.
 24. A crystal form of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-meth-oxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid having an X-ray powder diffraction pattern comprisingcharacteristic diffraction peaks at 6.56±0.2°, 13.20±0.2°, 19.86±0.2°,20.84±0.2°, 21.22±0.2°, and 25.22±0.2° 2θ.
 25. The crystal form of claim24, having an X-ray powder diffraction pattern comprising characteristicdiffraction peaks at 6.56±0.1°, 13.20±0.1°, 15.68±0.1°, 21.22±0.1°, and25.22±0.1° 2θ.
 26. The crystal form of claim 24, having an X-ray powderdiffraction pattern comprising characteristic diffraction peaks at6.56±0.06°, 13.20±0.06°, 15.68±0.06°, 20.84±0.06°, 21.22±0.06°, and25.22±0.06° 2θ.
 27. Crystal form I of6-(3-chloro-2-fluorobenzyl)-1-[(S)-1-hydroxymethyl-2-methylpropyl]-7-meth-oxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid having an X-ray powder diffraction pattern as shown in FIG.
 1. 28.The crystal form of any one of claims 15 to 27 having a purity ofcrystal of not less than 70%.
 29. The crystal form of any one of claims15 to 27 having a purity of crystal of not less than 80%.
 30. Thecrystal form of any one of claims 15 to 27 having a purity of crystal ofnot less than 90%.
 31. The crystal form of any one of claims 15 to 27having a purity of crystal of not less than 95%.
 32. The crystal form ofany one of claims 15 to 27 having a purity of crystal of not less than98%.
 33. A pharmaceutical composition comprising the crystal form of anyone of claims 15 to 27 and a pharmaceutically acceptable carrier. 34.The pharmaceutical composition of claim 33 wherein the composition is inthe form of a tablet, pill, powder or granule.